Antonie van Leeuwenhoek (2015) 107:1315–1321 DOI 10.1007/s10482-015-0426-2
ORIGINAL PAPER
Fontibacillus solani sp. nov. isolated from potato (Solanum tuberosum L.) root Martha-Helena Ramı´rez-Bahena • Jose´ David Flores-Fe´lix • Maria Jose´ Cuesta Carmen Tejedor Gil • Jose Luis Palomo • Pablo Garcı´a Benavides • Jose Mariano Igual • Mercedes Ferna´ndez Pascual • Encarna Vela´zquez • Alvaro Peix
•
Received: 16 December 2014 / Accepted: 5 March 2015 / Published online: 14 March 2015 Ó Springer International Publishing Switzerland 2015
Abstract A bacterial strain designated A4STR04T was isolated from the inner root tissue of potatoes in Spain. Phylogenetic analysis based on the 16S rRNA gene sequence placed the isolate into the genus Fontibacillus, being most closely related to Fontibacillus panacisegetis KCTC 13564T with 99 % identity. The isolate was observed to form Grampositive, motile and sporulating rods. The catalase test was found to be negative and oxidase positive. Nitrate was found to be reduced to nitrite. b-Galactosidase and caseinase were observed to be produced but the production of gelatinase, urease, arginine dehydrolase, ornithine and lysine decarboxylase was negative. Aesculin hydrolysis was found to be positive and acetoin production was negative. Growth was found to
Electronic supplementary material The online version of this article (doi:10.1007/s10482-015-0426-2) contains supplementary material, which is available to authorized users.
be supported by many carbohydrates and organic acids as carbon source. MK-7 was the only menaquinone detected and the major fatty acid (61.5 %) was identified as anteiso-C15:0, as occurs in the other species of genus Fontibacillus. The strain A4STR04T was found to display a complex lipid profile consisting of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, a glycolipid, two phospholipids, a lipid and two aminophospholipids. Mesodiaminopimelic acid was detected in the peptidoglycan. The G?C content was determined to be 50.5 mol% (Tm). Phylogenetic, chemotaxonomic and phenotypic analyses showed that strain A4STR04T (=LMG 28458 T = CECT 8693T) should be classified as representing a novel species of genus Fontibacillus, for which the name Fontibacillus solani sp. nov. is proposed. Keywords tuberosum
Fontibacillus Endophyte Solanum
M.-H. Ramı´rez-Bahena M. J. Cuesta J. M. Igual A. Peix (&) Instituto de Recursos Naturales y Agrobiologı´a de Salamanca, Consejo Superior de Investigaciones Cientı´ficas (IRNASA-CSIC), c/Cordel de Merinas 40-52, 37008 Salamanca, Spain e-mail: [email protected]; [email protected]
J. D. Flores-Fe´lix C. T. Gil E. Vela´zquez Departamento de Microbiologı´a y Gene´tica, Universidad de Salamanca, Salamanca, Spain
M.-H. Ramı´rez-Bahena J. M. Igual E. Vela´zquez A. Peix Unidad Asociada Grupo de Interaccio´n PlantaMicroorganismo, Universidad de Salamanca-IRNASACSIC, Salamanca, Spain
M. F. Pascual Instituto de Ciencias Agrarias ICA-CSIC, Madrid, Spain
J. L. Palomo P. G. Benavides Centro Regional de Diagno´stico, Junta de Castilla y Leo´n, Salamanca, Spain
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Introduction The genus Fontibacillus was described by Saha et al. (2010) and currently contains three species, Fontibacillus aquaticus, isolated from a warm spring, Fontibacillus panacisegetis, isolated from soil (Lee et al. 2011) and Fontibacillus phaseoli, isolated from nodules of Phaseolus vulgaris (Flores-Fe´lix et al. 2014). The genus belongs to the family Paenibacillaceae and comprises Gram-variable, motile and sporeforming rods with ellipsoidal terminal or subterminal endospores. The members of the genus are facultatively anaerobic and have menaquinone MK-7 as the major menaquinone. Their polar lipid profiles include phosphatidylglycerol, diphosphatidylglycerol and phosphatidylethanolamine as major lipids, and their major fatty acids are anteiso-C15:0, C16:0, isoC17:0 and anteiso-C17:0. The members of genus Fontibacillus are catalase negative and oxidase positive (Saha et al. 2010; Lee et al. 2011). In the present work we report a novel endophytic strain belonging to the genus Fontibacillus, A4STR04T, isolated from the inner root tissues of a potato (Solanum tuberosum) in Spain in the course of a project focused on the analysis of endophytic bacterial populations of this plant that constitutes one of the most important crops in this country. Phylogenetic and phenotypic, including chemotaxonomic, analyses showed that the strain A4STR04T belongs to a new species of the genus Fontibacillus for which the name Fontibacillus solani sp. nov. is proposed.
Materials and methods Bacterial isolation and culture The strain A4STR04T was isolated from the root of a plant of cultivar Laura growing in a soil located in Salamanca province (Spain). The isolation procedure was performed according to Aravind et al. (2009). Briefly, roots of potato plants were cut into 3.5 cm pieces, surface sterilized with 2 % sodium hypochlorite for 10 min and 70 % ethanol for a minute and rinsed five times in sterile distilled water. To check for reliability of the disinfection protocol and thus ensure the bacterial isolates came from the internal plant tissues (endophytes), 100 ll of the last wash water was spread on TSA medium plates to check for sterility.
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The disinfected root tissue samples were ground and macerated aseptically in phosphate buffered saline (PBS) (g l-1 NaCl 8, KCl 0.2, Na2HPO4 1.4 and KH2PO4 0.24, pH 7.4) and shaken at 200 rpm for 1 h at 28 °C. Serial dilutions of this suspension were spread on TSA plates and incubated at 28 °C for 7 days to yield colonies for purification. Subsequently, strain A4STR04T was grown on TSA medium (Difco, Becton Dickinson, BBL). Phenotypic tests Strain A4STR04T was grown on nutrient agar (NA) for 48 h at 22 °C to check for motility by phase-contrast microscopy using the hanging drop method. Gram staining was carried out by the procedure described by Doetsch (1981) after 24 h incubation at 28 °C. The flagellation type was determined by electron microscopy after 24 h incubation on NA at 22 °C. Cells were stained with 0.2 % uranyl acetate and examined at 80 kV with a STEM-LEO 910 transmission electron microscope (Zeiss, Oberkochen, Germany), equipped with a Gatan Bioscan 792 digital camera (Pleasanton, CA, USA). The phenotypic characterization was performed according to the standard methods as described by Claus and Berkeley (1986) and Logan and Berkeley (1984) and using the API 20NE, API20E, API50CH and API ZYM systems (bioMerieux, France) according to the manufacturer’s instructions. The results were recorded after 48 h incubation at 30 °C. The anaerobic growth was determined in liquid tetrathionate medium (Sigma, USA). Acetoin production, ability to grow in presence of 2, 5 and 7 % NaCl, nitrate reduction, phenylalanine deaminase, catalase, gelatinase, caseinase and oxidase were determined as described by Claus and Berkeley (1986). Acid production from D-glucose, D-xylose, D-mannitol and L-arabinose and gas from glucose were determined in liquid medium as described by Claus and Berkeley (1986). Growth was determined at temperatures ranging from 4, 15, 25, 37, 40 to 45 °C on TSA medium (Difco, Becton Dickinson, BBL). The growth at 45 °C was also tested in TSB (Difco, Becton Dickinson, BBL). The growth at pH 5.7 and 6.8 was tested as described Claus and Berkeley (1986) in Saboureaud broth without cloramphenicol (Difco, Becton Dickinson, BBL) and Nutrient broth (Difco, Becton Dickinson, BBL), respectively; the growth at
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pH 7–8 was tested in TSB medium containing 200 mM of Na2HPO4/NaH2PO4 and the growth at pH 9 and 10 was tested in the same medium containing 200 mM of NaHCO3/Na2CO3. The strains F. aquaticus DSM17643T, F. panacisegetis CECT 7605T and F. phaseoli BAPVE7BT were included in the phenotypic study as references.
Library). To perform the analysis of peptidoglycan, the whole cells of strain A4STR04T were hydrolysed with HCl at 100 °C for 15 h. The hydrolysates were subjected to thin-layer chromatography on cellulose plates using the solvent system of Rhuland et al. (1955) and the Protocol 1 of Schumann (2011).
Molecular studies
Results and discussion
For DNA base composition analysis, DNA was prepared according to Chun and Goodfellow (1995). The mol% G?C content of DNA was determined using the thermal denaturation method (Mandel and Marmur 1968). DNA–DNA hybridization was performed between strain A4STR04T and its closest relatives F. panacisegetis CECT 7605T, F. aquaticus DSM17643T and F. phaseoli BAPVE7BT by using the method of Ezaki et al. (1989), following the recommendations of Willems et al. (2001).
Strain A4STR04T was observed to form creamish white, round, smooth and convex colonies with approximate diameters of 1–3 mm on TSA plates after 24 h incubation at 28 °C. It was observed to be Gram-positive, to form oval central endospores in slightly swollen sporangia, and to be motile by means of peritrichous flagella (Supplementary Fig. S1). The comparison of the 16S rRNA gene sequence of A4STR04T (1486 nucleotides, Genbank accession number LN651195) with the sequences of type strains held in EzTaxon-e database indicated that this strain belongs to the genus Fontibacillus since its closest related species, with 99 % identity, was F. panacisegetis; the remaining species of the genus Fontibacillus, F. aquaticus and F. phaseoli presented less than 98 % identity. The NJ, ML and MP analyses showed similar results and are shown in Fig. 1. The G?C content of the strain A4STR04T was determined to be 50.5 mol%, which is a little higher than the values reported for other members of the genus Fontibacillus. DNA relatedness values between strain A4STR04T and its closest relatives F. panacisegetis CECT 7605T, F. aquaticus DSM17643T and F. phaseoli BAPVE7BT were 48 % (±8 %), 36 (±4 %) and 13 % (±2 %), respectively, confirming that the new isolate represents a new species of the genus Fontibacillus according to the current threshold for delineating prokaryotic species (Wayne et al. 1987). Menaquinone 7 (MK7) was the only respiratory quinone detected. This is also the predominant menaquinone in the other species of the genus Fontibacillus (Saha et al. 2010; Lee et al. 2011; Flores-Fe´lix et al. 2014). Mesodiaminopimelic acid (DAP) was detected in the peptidoglycan of strain A4STR04T, which presented peptidoglycan type A1c (Schumann 2011). The major fatty acid (61.5 %) was anteiso-C15:0 as occurs in the other species of Fontibacillus (Lee et al. 2011). The fatty acid profile also consisted of C14:0 (2.0 %); C15:0 (4.4 %); C16:0 (12.3 %); iso-C14:0
Phylogenetic analysis Amplification and sequencing of the 16S rRNA gene were performed according to Rivas et al. (2007). The sequence obtained was compared with those from the GenBank using BLASTN (Altschul et al. 1990) and EzTaxon-e server (Kim et al. 2012). Sequences were aligned using the Clustal_X software (Thompson et al. 1997) and distances were calculated according to Kimura0 s two-parameter model (Kimura 1980). The phylogenetic trees were inferred using the neighbour joining (NJ), maximum likelihood (ML) and maximum parsimony (MP) models (Saitou and Nei 1987; Rogers and Swofford 1998; Felsenstein 1983). MEGA5 package (Tamura et al. 2011) was used for all analyses. Chemotaxonomic analyses The chemotaxonomic analyses were carried out by the Identification Service of DSMZ (Braunschweig, Germany). Isolate A4STR04T was cultivated on TSA (Becton Dickinson, BBL) for 48 h at 28 °C. The respiratory quinones and polar lipids were analysed as described by Tindall (1990) and the cellular fatty acids according to the instructions of the Microbial Identification System (MIDI; Microbial ID; TSBA40 4.0
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100
58
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96 Fontibacillus solani A4STR04T (LN651195 ) Fontibacillus panacisegetis KCTC 13564T (GQ303568) Fontibacillus phaseoli BAPVE7BT (KF583881) Fontibacillus aquaticus GPTSA 19T (DQ023221) Paenibacillus polymyxa DSM 36T (AJ320493) Cohnella thermotolerans CCUG 47242T (AJ971483) Thermobacillus xylanilyticus XET (AJ005795) 100 Ammoniphilus oxalaticus RAOx1T (Y14578) Oxalophagus oxalicus DSM 5503T (Y14581) Aneurinibacillus aneurinolyticus DSM 5562T (X94194) Brevibacillus brevis NBRC 15304T (AB271756)
0.02
Fig. 1 Maximun likelihood phylogenetic tree based on the nearly complete 16S rRNA gene sequence (1482 nucleotides) showing the phylogenetic position of Fontibacillus solani A4STR04T within the family Paenibacillaceae. Only bootstrap values[50 % (percentage of 1000 replications) are shown at the
nodes. Filled circles indicate nodes that were also found with the neighbour-joining and maximum parsimony algorithms. Filled squares indicate nodes that were found with neighbour-joining and maximum parsimony algorithms. Bar 2 nt substitutions per 100 nt
Table 1 Cellular fatty acid composition of strain A4STR04T and related type strains of Fontibacillus species (all data were obtained in this study) Fatty acid
F. solani A4STR04T
F. panacisegetis CECT 7605T
F. phaseoli BAPVE7BT
F. aquaticus DSM 17643T
Saturated straight-chain C12:0
0.5
0.6
1.1
0.7
C14:0
2.0
4.6
3.3
6.0
C15:0
4.4
5.0
4.1
4.4
C16:0
12.3
13.4
18.1
21.3
Saturated iso-branched C14:0
1.3
2.0
1.5
1.2
C15:0
3.6
2.4
2.8
3.9
C16:0
5.1
3.3
4.2
4.8
C17:0
1.2
0.3
1.0
1.0
nd
0.9
Saturated anteiso-branched C13:0
0.7
1.4
C15:0
61.5
58.5
55.8
50.5
C17:0
6.8
3.0
5.7
3.5
nd
4.4
0.7
nd
Unsaturated C16:1x11c
Fatty acids present in amounts lower than 1 % in all species are not shown nd not detected
(1.3 %); iso-C15:0 (3.6 %); iso-C16:0 (5.1 %); iso-C17:0 (1.2 %) and anteiso-C17:0 (6.8 %) and several fatty acids in amounts lower than 1 %. The fatty acid profile of strain A4STR04T is similar to those of the reference Fontibacillus species and only slight differences were found in the amounts of some fatty acids (Table 1). The strain A4STR04T was found to display a complex lipid profile (Supplementary Fig. S2) consisting of
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diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, a glycolipid, two phospholipids, a lipid and two aminophospholipids. This profile is similar to those of other Fontibacillus species since, from the identified polar lipids, diphosphatidylglycerol and phosphatidylglycerol were found in all species and phosphatidylethanolamine is present in most species, including the novel species proposed
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Table 2 Differential phenotypic characteristics of Fontibacillus solani sp. nov. and the other Fontibacillus species Characteristics
F. solani A4STR04T
F. panacisegetis CECT 7605T
F. phaseoli BAPVE7BT
F. aquaticus DSM 17643T
Gram reaction
?
-
?
?
Nitrate reduction
?
?
?
-
Caseinase
?
w
-
-
Voges-Proskauer
-
?
?
-
?
-
?
-
N-acetylglucosamine
-
-
-
?
Gluconate
-
-
?
-
-
?
-
?
Enzymatic activity (in API ZYM) Acid phosphatase Assimilation of (in API 20NE)
Acid from (in API 50CH) Glycerol Erythritol
-
?
-
?
L-arabinose
-
?
-
?
D-ribose
-
-
-
?
Fructose D-mannose
? w
? ?
-
? ?
Melibiose
?
-
?
-
Sucrose
w
-
?
-
Trehalose
w
-
?
-
L-lyxose
-
-
?
-
C15:0 anteiso
61.5
58.5
55.8
50.5
C16:0
12.3
13.4
18.1
21.3
Identified polar lipids
PE
PE
not detected
PE
Unidentified polar lipids
not detected
APGL
AL1, AL2,
AL2, AL3, APGL
Major fatty acids (percentage)
? Positive reaction, - negative reaction, w weakly positive reaction PE phosphatidylethanolamine, AL aminolipid, APGL aminophosphoglycolipid
in this study (Saha et al. 2010; Lee et al. 2011; Flores-Fe´lix et al. 2014). Regarding the unknown polar lipids in strain A4STR04T and in the remaining species of the genus Fontibacillus, lipids, glycolipids, phospholipids and aminophospholipids were found, with aminophosphoglycolipids present only in F. aquaticus and F. panacisegetis and aminolipids only present in the type species of the genus, F. aquaticus (Table 2). The phenotypic characterization included the characteristics recommended in the minimal standards for aerobic endospore-forming bacteria (Logan et al. 2009). The results are given in the species description below and the differences compared with the related species are summarised in Table 2.
The phylogenetic, chemotaxonomic and phenotypic analyses showed that the strain A4STR04T represents a new species of the genus Fontibacillus for which the name Fontibacillus solani sp. nov. is proposed.
Description of Fontibacillus solani sp. nov Fontibacillus solani (so.la’ni. N.L. gen. neut. n. solani of the potato, the isolation source of this species) Cells are Gram-positive rods (width 0.4–0.6 lm, length 1.7–2.0 lm) and motile by means of
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peritrichous flagella. Oval central endospores are formed in slightly swollen sporangia. Catalase negative and oxidase positive. Colonies on NA medium are creamish white, round, smooth and convex with approximate diameters of 1–3 mm. Anaerobic growth is positive. Grows at pH 8 and weakly at pH 5.7, but not at pH 9 and the optimal pH is 7. Grows in the presence of 2 % NaCl but not in 5 % NaCl and the optimal NaCl concentration for growth is 0.5–1 %. Grows at 40 °C but not at 10 or 45 °C and the optimal temperature for growth is 30 °C. Menaquinone 7 (MK7) is the only respiratory quinone and DAP is present in the peptidoglycan. The fatty acid profile contains anteiso-C15:0 as the major fatty acid and also contains C14:0, C15:0, C16:0, iso-C14:0, iso-C15:0, isoC16:0, iso-C17:0 and anteiso-C17:0. The polar lipid profile consists of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, a glycolipid, two phospholipids, one lipid, and two aminophospholipids. Nitrate is reduced to nitrite. Produces b-galactosidase and caseinase but does not produce gelatinase, indole, phenylalanine deaminase, urease, arginine dehydrolase, ornithine or lysine decarboxylase. H2S production is negative. Aesculin hydrolysis is positive and Voges-Proskauer is negative. In the API 20NE system the assimilation of glucose, mannose and maltose is positive and that of Larabinose, mannitol, N-acetyl-glucosamine, gluconate, caprate, adipate, malate, citrate and phenylacetate is negative. Acid but not gas is produced from glucose. In the API 50CH tests, the acid production from glucose, fructose, amygdalin, arbutin, salicin, cellobiose, maltose, lactose, melibiose, starch, glycogen and gentiobiose is positive. The acid production from glycerol, erythritol, D-arabinose, L-arabinose, D-ribose, D-xylose, L-xylose, adonitol, methyl-a-Dxyloside, L-sorbose, L-rhamnose, dulcitol, inositol, mannitol, sorbitol, methyl-a-D-mannoside, N-acetylglucosamine, inulin, melezitose, turanose, L-lyxose, tagatose, D-fucose, L-fucose, D-arabinitol and L-arabinitol is negative. The acid production from galactose, D-mannose, methyl-a-D-glucoside, sucrose, raffinose, trehalose and xylitol is weak. The hydrolysis of aesculin is positive. Assimilation of gluconate and 2- and 5-Keto-gluconate is negative. In the API ZYM system, leucine arylamidase, chemotrypsin, acid and alkaline phosphatases, phosphohydrolase, a and b glucosidases, and a-galactosidase are produced in addition to b-galactosidase. Lipase, esterase, esterase
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lipase, valine and cystine arylamidases, trypsin, glucuronidase, N-acetyl-glucosaminidase, a-mannosidase and a-fucosidase production is negative. The G?C content of the type strain is 50.5 mol% (Tm). The type strain A4STR04T (=LMG 28458T, = CECT 8693T) was isolated from a S. tuberosum root in Spain. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain A4STR04T is LN651195. Acknowledgments This research was funded by Junta de Castilla y Leo´n (Spanish Regional Government) under project CSI327A12-2. MHRB is recipient of a JAE-Doc researcher contract from CSIC cofinanced by ERDF. We are grateful to IDEBIO S. L. and V. Souza-Egipsy from ICA Electron Microscopy Service. Conflict of interest The authors declare that they have no conflict of interest. Ethical standard This article does not contain any studies with human participants or animals performed by any of the authors.
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ORIGINAL PAPER
Fontibacillus solani sp. nov. isolated from potato (Solanum tuberosum L.) root Martha-Helena Ramı´rez-Bahena • Jose´ David Flores-Fe´lix • Maria Jose´ Cuesta Carmen Tejedor Gil • Jose Luis Palomo • Pablo Garcı´a Benavides • Jose Mariano Igual • Mercedes Ferna´ndez Pascual • Encarna Vela´zquez • Alvaro Peix
•
Received: 16 December 2014 / Accepted: 5 March 2015 / Published online: 14 March 2015 Ó Springer International Publishing Switzerland 2015
Abstract A bacterial strain designated A4STR04T was isolated from the inner root tissue of potatoes in Spain. Phylogenetic analysis based on the 16S rRNA gene sequence placed the isolate into the genus Fontibacillus, being most closely related to Fontibacillus panacisegetis KCTC 13564T with 99 % identity. The isolate was observed to form Grampositive, motile and sporulating rods. The catalase test was found to be negative and oxidase positive. Nitrate was found to be reduced to nitrite. b-Galactosidase and caseinase were observed to be produced but the production of gelatinase, urease, arginine dehydrolase, ornithine and lysine decarboxylase was negative. Aesculin hydrolysis was found to be positive and acetoin production was negative. Growth was found to
Electronic supplementary material The online version of this article (doi:10.1007/s10482-015-0426-2) contains supplementary material, which is available to authorized users.
be supported by many carbohydrates and organic acids as carbon source. MK-7 was the only menaquinone detected and the major fatty acid (61.5 %) was identified as anteiso-C15:0, as occurs in the other species of genus Fontibacillus. The strain A4STR04T was found to display a complex lipid profile consisting of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, a glycolipid, two phospholipids, a lipid and two aminophospholipids. Mesodiaminopimelic acid was detected in the peptidoglycan. The G?C content was determined to be 50.5 mol% (Tm). Phylogenetic, chemotaxonomic and phenotypic analyses showed that strain A4STR04T (=LMG 28458 T = CECT 8693T) should be classified as representing a novel species of genus Fontibacillus, for which the name Fontibacillus solani sp. nov. is proposed. Keywords tuberosum
Fontibacillus Endophyte Solanum
M.-H. Ramı´rez-Bahena M. J. Cuesta J. M. Igual A. Peix (&) Instituto de Recursos Naturales y Agrobiologı´a de Salamanca, Consejo Superior de Investigaciones Cientı´ficas (IRNASA-CSIC), c/Cordel de Merinas 40-52, 37008 Salamanca, Spain e-mail: [email protected]; [email protected]
J. D. Flores-Fe´lix C. T. Gil E. Vela´zquez Departamento de Microbiologı´a y Gene´tica, Universidad de Salamanca, Salamanca, Spain
M.-H. Ramı´rez-Bahena J. M. Igual E. Vela´zquez A. Peix Unidad Asociada Grupo de Interaccio´n PlantaMicroorganismo, Universidad de Salamanca-IRNASACSIC, Salamanca, Spain
M. F. Pascual Instituto de Ciencias Agrarias ICA-CSIC, Madrid, Spain
J. L. Palomo P. G. Benavides Centro Regional de Diagno´stico, Junta de Castilla y Leo´n, Salamanca, Spain
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Introduction The genus Fontibacillus was described by Saha et al. (2010) and currently contains three species, Fontibacillus aquaticus, isolated from a warm spring, Fontibacillus panacisegetis, isolated from soil (Lee et al. 2011) and Fontibacillus phaseoli, isolated from nodules of Phaseolus vulgaris (Flores-Fe´lix et al. 2014). The genus belongs to the family Paenibacillaceae and comprises Gram-variable, motile and sporeforming rods with ellipsoidal terminal or subterminal endospores. The members of the genus are facultatively anaerobic and have menaquinone MK-7 as the major menaquinone. Their polar lipid profiles include phosphatidylglycerol, diphosphatidylglycerol and phosphatidylethanolamine as major lipids, and their major fatty acids are anteiso-C15:0, C16:0, isoC17:0 and anteiso-C17:0. The members of genus Fontibacillus are catalase negative and oxidase positive (Saha et al. 2010; Lee et al. 2011). In the present work we report a novel endophytic strain belonging to the genus Fontibacillus, A4STR04T, isolated from the inner root tissues of a potato (Solanum tuberosum) in Spain in the course of a project focused on the analysis of endophytic bacterial populations of this plant that constitutes one of the most important crops in this country. Phylogenetic and phenotypic, including chemotaxonomic, analyses showed that the strain A4STR04T belongs to a new species of the genus Fontibacillus for which the name Fontibacillus solani sp. nov. is proposed.
Materials and methods Bacterial isolation and culture The strain A4STR04T was isolated from the root of a plant of cultivar Laura growing in a soil located in Salamanca province (Spain). The isolation procedure was performed according to Aravind et al. (2009). Briefly, roots of potato plants were cut into 3.5 cm pieces, surface sterilized with 2 % sodium hypochlorite for 10 min and 70 % ethanol for a minute and rinsed five times in sterile distilled water. To check for reliability of the disinfection protocol and thus ensure the bacterial isolates came from the internal plant tissues (endophytes), 100 ll of the last wash water was spread on TSA medium plates to check for sterility.
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The disinfected root tissue samples were ground and macerated aseptically in phosphate buffered saline (PBS) (g l-1 NaCl 8, KCl 0.2, Na2HPO4 1.4 and KH2PO4 0.24, pH 7.4) and shaken at 200 rpm for 1 h at 28 °C. Serial dilutions of this suspension were spread on TSA plates and incubated at 28 °C for 7 days to yield colonies for purification. Subsequently, strain A4STR04T was grown on TSA medium (Difco, Becton Dickinson, BBL). Phenotypic tests Strain A4STR04T was grown on nutrient agar (NA) for 48 h at 22 °C to check for motility by phase-contrast microscopy using the hanging drop method. Gram staining was carried out by the procedure described by Doetsch (1981) after 24 h incubation at 28 °C. The flagellation type was determined by electron microscopy after 24 h incubation on NA at 22 °C. Cells were stained with 0.2 % uranyl acetate and examined at 80 kV with a STEM-LEO 910 transmission electron microscope (Zeiss, Oberkochen, Germany), equipped with a Gatan Bioscan 792 digital camera (Pleasanton, CA, USA). The phenotypic characterization was performed according to the standard methods as described by Claus and Berkeley (1986) and Logan and Berkeley (1984) and using the API 20NE, API20E, API50CH and API ZYM systems (bioMerieux, France) according to the manufacturer’s instructions. The results were recorded after 48 h incubation at 30 °C. The anaerobic growth was determined in liquid tetrathionate medium (Sigma, USA). Acetoin production, ability to grow in presence of 2, 5 and 7 % NaCl, nitrate reduction, phenylalanine deaminase, catalase, gelatinase, caseinase and oxidase were determined as described by Claus and Berkeley (1986). Acid production from D-glucose, D-xylose, D-mannitol and L-arabinose and gas from glucose were determined in liquid medium as described by Claus and Berkeley (1986). Growth was determined at temperatures ranging from 4, 15, 25, 37, 40 to 45 °C on TSA medium (Difco, Becton Dickinson, BBL). The growth at 45 °C was also tested in TSB (Difco, Becton Dickinson, BBL). The growth at pH 5.7 and 6.8 was tested as described Claus and Berkeley (1986) in Saboureaud broth without cloramphenicol (Difco, Becton Dickinson, BBL) and Nutrient broth (Difco, Becton Dickinson, BBL), respectively; the growth at
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pH 7–8 was tested in TSB medium containing 200 mM of Na2HPO4/NaH2PO4 and the growth at pH 9 and 10 was tested in the same medium containing 200 mM of NaHCO3/Na2CO3. The strains F. aquaticus DSM17643T, F. panacisegetis CECT 7605T and F. phaseoli BAPVE7BT were included in the phenotypic study as references.
Library). To perform the analysis of peptidoglycan, the whole cells of strain A4STR04T were hydrolysed with HCl at 100 °C for 15 h. The hydrolysates were subjected to thin-layer chromatography on cellulose plates using the solvent system of Rhuland et al. (1955) and the Protocol 1 of Schumann (2011).
Molecular studies
Results and discussion
For DNA base composition analysis, DNA was prepared according to Chun and Goodfellow (1995). The mol% G?C content of DNA was determined using the thermal denaturation method (Mandel and Marmur 1968). DNA–DNA hybridization was performed between strain A4STR04T and its closest relatives F. panacisegetis CECT 7605T, F. aquaticus DSM17643T and F. phaseoli BAPVE7BT by using the method of Ezaki et al. (1989), following the recommendations of Willems et al. (2001).
Strain A4STR04T was observed to form creamish white, round, smooth and convex colonies with approximate diameters of 1–3 mm on TSA plates after 24 h incubation at 28 °C. It was observed to be Gram-positive, to form oval central endospores in slightly swollen sporangia, and to be motile by means of peritrichous flagella (Supplementary Fig. S1). The comparison of the 16S rRNA gene sequence of A4STR04T (1486 nucleotides, Genbank accession number LN651195) with the sequences of type strains held in EzTaxon-e database indicated that this strain belongs to the genus Fontibacillus since its closest related species, with 99 % identity, was F. panacisegetis; the remaining species of the genus Fontibacillus, F. aquaticus and F. phaseoli presented less than 98 % identity. The NJ, ML and MP analyses showed similar results and are shown in Fig. 1. The G?C content of the strain A4STR04T was determined to be 50.5 mol%, which is a little higher than the values reported for other members of the genus Fontibacillus. DNA relatedness values between strain A4STR04T and its closest relatives F. panacisegetis CECT 7605T, F. aquaticus DSM17643T and F. phaseoli BAPVE7BT were 48 % (±8 %), 36 (±4 %) and 13 % (±2 %), respectively, confirming that the new isolate represents a new species of the genus Fontibacillus according to the current threshold for delineating prokaryotic species (Wayne et al. 1987). Menaquinone 7 (MK7) was the only respiratory quinone detected. This is also the predominant menaquinone in the other species of the genus Fontibacillus (Saha et al. 2010; Lee et al. 2011; Flores-Fe´lix et al. 2014). Mesodiaminopimelic acid (DAP) was detected in the peptidoglycan of strain A4STR04T, which presented peptidoglycan type A1c (Schumann 2011). The major fatty acid (61.5 %) was anteiso-C15:0 as occurs in the other species of Fontibacillus (Lee et al. 2011). The fatty acid profile also consisted of C14:0 (2.0 %); C15:0 (4.4 %); C16:0 (12.3 %); iso-C14:0
Phylogenetic analysis Amplification and sequencing of the 16S rRNA gene were performed according to Rivas et al. (2007). The sequence obtained was compared with those from the GenBank using BLASTN (Altschul et al. 1990) and EzTaxon-e server (Kim et al. 2012). Sequences were aligned using the Clustal_X software (Thompson et al. 1997) and distances were calculated according to Kimura0 s two-parameter model (Kimura 1980). The phylogenetic trees were inferred using the neighbour joining (NJ), maximum likelihood (ML) and maximum parsimony (MP) models (Saitou and Nei 1987; Rogers and Swofford 1998; Felsenstein 1983). MEGA5 package (Tamura et al. 2011) was used for all analyses. Chemotaxonomic analyses The chemotaxonomic analyses were carried out by the Identification Service of DSMZ (Braunschweig, Germany). Isolate A4STR04T was cultivated on TSA (Becton Dickinson, BBL) for 48 h at 28 °C. The respiratory quinones and polar lipids were analysed as described by Tindall (1990) and the cellular fatty acids according to the instructions of the Microbial Identification System (MIDI; Microbial ID; TSBA40 4.0
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87
94
98
100
58
52
96 Fontibacillus solani A4STR04T (LN651195 ) Fontibacillus panacisegetis KCTC 13564T (GQ303568) Fontibacillus phaseoli BAPVE7BT (KF583881) Fontibacillus aquaticus GPTSA 19T (DQ023221) Paenibacillus polymyxa DSM 36T (AJ320493) Cohnella thermotolerans CCUG 47242T (AJ971483) Thermobacillus xylanilyticus XET (AJ005795) 100 Ammoniphilus oxalaticus RAOx1T (Y14578) Oxalophagus oxalicus DSM 5503T (Y14581) Aneurinibacillus aneurinolyticus DSM 5562T (X94194) Brevibacillus brevis NBRC 15304T (AB271756)
0.02
Fig. 1 Maximun likelihood phylogenetic tree based on the nearly complete 16S rRNA gene sequence (1482 nucleotides) showing the phylogenetic position of Fontibacillus solani A4STR04T within the family Paenibacillaceae. Only bootstrap values[50 % (percentage of 1000 replications) are shown at the
nodes. Filled circles indicate nodes that were also found with the neighbour-joining and maximum parsimony algorithms. Filled squares indicate nodes that were found with neighbour-joining and maximum parsimony algorithms. Bar 2 nt substitutions per 100 nt
Table 1 Cellular fatty acid composition of strain A4STR04T and related type strains of Fontibacillus species (all data were obtained in this study) Fatty acid
F. solani A4STR04T
F. panacisegetis CECT 7605T
F. phaseoli BAPVE7BT
F. aquaticus DSM 17643T
Saturated straight-chain C12:0
0.5
0.6
1.1
0.7
C14:0
2.0
4.6
3.3
6.0
C15:0
4.4
5.0
4.1
4.4
C16:0
12.3
13.4
18.1
21.3
Saturated iso-branched C14:0
1.3
2.0
1.5
1.2
C15:0
3.6
2.4
2.8
3.9
C16:0
5.1
3.3
4.2
4.8
C17:0
1.2
0.3
1.0
1.0
nd
0.9
Saturated anteiso-branched C13:0
0.7
1.4
C15:0
61.5
58.5
55.8
50.5
C17:0
6.8
3.0
5.7
3.5
nd
4.4
0.7
nd
Unsaturated C16:1x11c
Fatty acids present in amounts lower than 1 % in all species are not shown nd not detected
(1.3 %); iso-C15:0 (3.6 %); iso-C16:0 (5.1 %); iso-C17:0 (1.2 %) and anteiso-C17:0 (6.8 %) and several fatty acids in amounts lower than 1 %. The fatty acid profile of strain A4STR04T is similar to those of the reference Fontibacillus species and only slight differences were found in the amounts of some fatty acids (Table 1). The strain A4STR04T was found to display a complex lipid profile (Supplementary Fig. S2) consisting of
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diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, a glycolipid, two phospholipids, a lipid and two aminophospholipids. This profile is similar to those of other Fontibacillus species since, from the identified polar lipids, diphosphatidylglycerol and phosphatidylglycerol were found in all species and phosphatidylethanolamine is present in most species, including the novel species proposed
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Table 2 Differential phenotypic characteristics of Fontibacillus solani sp. nov. and the other Fontibacillus species Characteristics
F. solani A4STR04T
F. panacisegetis CECT 7605T
F. phaseoli BAPVE7BT
F. aquaticus DSM 17643T
Gram reaction
?
-
?
?
Nitrate reduction
?
?
?
-
Caseinase
?
w
-
-
Voges-Proskauer
-
?
?
-
?
-
?
-
N-acetylglucosamine
-
-
-
?
Gluconate
-
-
?
-
-
?
-
?
Enzymatic activity (in API ZYM) Acid phosphatase Assimilation of (in API 20NE)
Acid from (in API 50CH) Glycerol Erythritol
-
?
-
?
L-arabinose
-
?
-
?
D-ribose
-
-
-
?
Fructose D-mannose
? w
? ?
-
? ?
Melibiose
?
-
?
-
Sucrose
w
-
?
-
Trehalose
w
-
?
-
L-lyxose
-
-
?
-
C15:0 anteiso
61.5
58.5
55.8
50.5
C16:0
12.3
13.4
18.1
21.3
Identified polar lipids
PE
PE
not detected
PE
Unidentified polar lipids
not detected
APGL
AL1, AL2,
AL2, AL3, APGL
Major fatty acids (percentage)
? Positive reaction, - negative reaction, w weakly positive reaction PE phosphatidylethanolamine, AL aminolipid, APGL aminophosphoglycolipid
in this study (Saha et al. 2010; Lee et al. 2011; Flores-Fe´lix et al. 2014). Regarding the unknown polar lipids in strain A4STR04T and in the remaining species of the genus Fontibacillus, lipids, glycolipids, phospholipids and aminophospholipids were found, with aminophosphoglycolipids present only in F. aquaticus and F. panacisegetis and aminolipids only present in the type species of the genus, F. aquaticus (Table 2). The phenotypic characterization included the characteristics recommended in the minimal standards for aerobic endospore-forming bacteria (Logan et al. 2009). The results are given in the species description below and the differences compared with the related species are summarised in Table 2.
The phylogenetic, chemotaxonomic and phenotypic analyses showed that the strain A4STR04T represents a new species of the genus Fontibacillus for which the name Fontibacillus solani sp. nov. is proposed.
Description of Fontibacillus solani sp. nov Fontibacillus solani (so.la’ni. N.L. gen. neut. n. solani of the potato, the isolation source of this species) Cells are Gram-positive rods (width 0.4–0.6 lm, length 1.7–2.0 lm) and motile by means of
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peritrichous flagella. Oval central endospores are formed in slightly swollen sporangia. Catalase negative and oxidase positive. Colonies on NA medium are creamish white, round, smooth and convex with approximate diameters of 1–3 mm. Anaerobic growth is positive. Grows at pH 8 and weakly at pH 5.7, but not at pH 9 and the optimal pH is 7. Grows in the presence of 2 % NaCl but not in 5 % NaCl and the optimal NaCl concentration for growth is 0.5–1 %. Grows at 40 °C but not at 10 or 45 °C and the optimal temperature for growth is 30 °C. Menaquinone 7 (MK7) is the only respiratory quinone and DAP is present in the peptidoglycan. The fatty acid profile contains anteiso-C15:0 as the major fatty acid and also contains C14:0, C15:0, C16:0, iso-C14:0, iso-C15:0, isoC16:0, iso-C17:0 and anteiso-C17:0. The polar lipid profile consists of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, a glycolipid, two phospholipids, one lipid, and two aminophospholipids. Nitrate is reduced to nitrite. Produces b-galactosidase and caseinase but does not produce gelatinase, indole, phenylalanine deaminase, urease, arginine dehydrolase, ornithine or lysine decarboxylase. H2S production is negative. Aesculin hydrolysis is positive and Voges-Proskauer is negative. In the API 20NE system the assimilation of glucose, mannose and maltose is positive and that of Larabinose, mannitol, N-acetyl-glucosamine, gluconate, caprate, adipate, malate, citrate and phenylacetate is negative. Acid but not gas is produced from glucose. In the API 50CH tests, the acid production from glucose, fructose, amygdalin, arbutin, salicin, cellobiose, maltose, lactose, melibiose, starch, glycogen and gentiobiose is positive. The acid production from glycerol, erythritol, D-arabinose, L-arabinose, D-ribose, D-xylose, L-xylose, adonitol, methyl-a-Dxyloside, L-sorbose, L-rhamnose, dulcitol, inositol, mannitol, sorbitol, methyl-a-D-mannoside, N-acetylglucosamine, inulin, melezitose, turanose, L-lyxose, tagatose, D-fucose, L-fucose, D-arabinitol and L-arabinitol is negative. The acid production from galactose, D-mannose, methyl-a-D-glucoside, sucrose, raffinose, trehalose and xylitol is weak. The hydrolysis of aesculin is positive. Assimilation of gluconate and 2- and 5-Keto-gluconate is negative. In the API ZYM system, leucine arylamidase, chemotrypsin, acid and alkaline phosphatases, phosphohydrolase, a and b glucosidases, and a-galactosidase are produced in addition to b-galactosidase. Lipase, esterase, esterase
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lipase, valine and cystine arylamidases, trypsin, glucuronidase, N-acetyl-glucosaminidase, a-mannosidase and a-fucosidase production is negative. The G?C content of the type strain is 50.5 mol% (Tm). The type strain A4STR04T (=LMG 28458T, = CECT 8693T) was isolated from a S. tuberosum root in Spain. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain A4STR04T is LN651195. Acknowledgments This research was funded by Junta de Castilla y Leo´n (Spanish Regional Government) under project CSI327A12-2. MHRB is recipient of a JAE-Doc researcher contract from CSIC cofinanced by ERDF. We are grateful to IDEBIO S. L. and V. Souza-Egipsy from ICA Electron Microscopy Service. Conflict of interest The authors declare that they have no conflict of interest. Ethical standard This article does not contain any studies with human participants or animals performed by any of the authors.
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