International Journal of Systematic and Evolutionary Microbiology (2015), 65, 2542–2548 DOI 10.1099/ijs.0.000298

Sphingomonas zeae sp. nov., isolated from the stem of Zea mays

Peter Ka¨mpfer,1 Hans-Ju¨rgen Busse,2 John A. McInroy3 and Stefanie P. Glaeser1

Correspondence 1Institut fu¨r Angewandte Mikrobiologie, Universita¨t Giessen, Giessen, Germany Peter Ka¨mpfer 2Institut fu¨r Mikrobiologie, Veterina¨rmedizinische Universita¨t, A-1210 Wien, Austria peter.kaempfer@agrar. 3 uni-giessen.de Department of Entomology and Plant Pathology, Auburn University, Alabama, USA

A yellow-pigmented bacterial isolate (strain JM-791T) obtained from the healthy internal stem tissue of 1-month-old corn (Zea mays, cultivar ‘Sweet Belle’) grown at the Plant Breeding Unit of the E.V. Smith Research Center in Tallassee (Elmore county), Alabama, USA, was taxonomically characterized. The study employing a polyphasic approach, including 16S RNA gene sequence analysis, physiological characterization, estimation of the ubiquinone and polar lipid patterns, and fatty acid composition, revealed that strain JM-791T shared 16S rRNA gene sequence similarities with type strains of paucimobilis (98.3 %), Sphingomonas pseudosanguinis (97.5 %) and Sphingomonas yabuuchiae (97.4 %), but also showed pronounced differences, both genotypically and phenotypically. On the basis of these results, a novel species of the genus Sphingomonas is described, for which we propose the name Sphingomonas zeae sp. nov. with the type strain JM-791T (5LMG 28739T5CCM 8596T).

The genus Sphingomonas, proposed by Yabuuchi et al. Strain JM-791T was originally grown on Tryptic Soy Agar (1990) encompassed Gram-negative, non-fermentative (TSA; Oxoid), and was further studied and sub-cultivated rods, which can be chemotaxonomically characterized by on nutrient agar (NA; Oxoid) at 28 8C for 48 h and sub- the presence of ubiquinone Q-10, sym-homospermidine sequently analysed for its 16S rRNA gene sequence, fatty as the key polyamine, a lipid pattern consisting of phospha- acid methyl ester composition of the whole-cell hydroly- sate, further phenotypic features, and DNA–DNA related- tidylethanolamine (exception: T ness to those species most closely related on the basis of DSM 1805 ; Denner et al., 1999), phosphatidylglycerol, 16S rRNA gene sequence similarities. diphosphatidylglycerol, sphingoglycolipid and phospha- 8 tidylcholine as major lipids, and the presence of 2-hydroxy- On NA at 28 C the strain showed a yellow pigmentation. Cells of strain JM-791T stained as Gram-negative with the myristic acid (C 2-OH) and the absence of 3-hydroxy 14 : 0 modified Hucker method after Gerhardt et al. (1994). fatty acids in their fatty acid profiles (Busse et al., 1999; Cell morphology was observed under a Zeiss light micro- Takeuchi et al., 2001; Zhang et al., 2005; Yoon et al., 2006). scope at 61000 magnification, with cells grown for 24 h Members of the genus have been isolated from roots or rhi- at 28 8C on medium NA. Good growth occurred on NA, zosphere soil (Xie & Yokoto, 2006; Takeuchi et al., 1995; as well as on brain heart infusion agar, R2A agar and Chung et al., 2011), the phyllosphere (Tala` et al., 2013; TSA, but no growth was observed on MacConkey agar Rivas et al., 2004) and also from endophytic compartments (Oxoid). Very reduced growth was observed at 4 8C and of plants (Huang et al., 2012) suggesting an important role 45 8C. The best growth was recorded at temperatures 8 8 T of the genus for plant–microbe interaction. between 25 C and 30 C. Strain JM-791 grew in TSB at 28 8C in the presence of 1 %, but not 2 % (w/v) NaCl In this study employing a polyphasic characterization, we and above, and in TSB adjusted to pH 5.5–9.5 but not at describe a novel species of the genus Sphingomonas, which pH 4.5 or pH 10.5. was isolated from the healthy internal stem tissue of 1- Detailed physiological characterization and biochemical month-old corn (Zea mays, cultivar ‘Sweet Belle’) grown at tests were performed to assess the carbon source utilization the Plant Breeding Unit of the E.V. Smith Research Center pattern, acid formation from different sugars and/or sugar- in Tallassee (Elmore county), Alabama, USA. related compounds, and hydrolysis of chromogenic sub- strates as described by Ka¨mpfer et al. (1991). In addition, The GenBank/EMBL/DDBJ accession number for the 16S rRNA other biochemical tests were performed, such as pro- gene sequence of strain JM-791T is KP999966. duction of hydrogen sulphide, indole reaction with

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Ehrlich’s and Kovacs’ reagents, the activity of arginine and tyrosine were performed according to Smibert & dihydrolase, lysine decarboxylase, ornithine decarboxylase, Krieg (1994). Isolate JM-791T utilized many carbon b-galactosidase (ONPG), and urease on Christensen’s urea sources, similar to all species of the genus Sphingomonas, agar (all performed with the Micronaut E kit; Ka¨mpfer, and was able to produce acid from D-glucose, lactose 1990). Hydrolysis of casein, gelatin (plate method), starch (weak), sucrose, L-arabinose (weak), maltose, D-xylose,

Table 1. Differentiating characteristics of strain JM-791T, and related sym-homospermidine-containing species of the genus Sphingomonas

1, JM-791T;2,S. pseudosanguinis G1-2T;3,S. yabuuchiae DSM 14562T;4,S. sanguinis NBRC 13937T;5,S. pituitosa EDIVT;6,S. trueperi ATCC 12417T;7,S. paucimobilis ATCC 29837T;8,S. parapaucimobilis JCM 7510T;9,S. roseiflava IAM 14823T. Data for taxa 1 from this study; data for taxa 2–9 from Ka¨mpfer et al. (2007), obtained under exactly the same conditions. All data were obtained with the same method (Ka¨mpfer et al., 1991).+, Positive; (+), weakly positive; 2, negative.

Characteristic 1 2 3 4 5 6 7 8 9

Acid production from: Glucose + 2 (+) 2 (+) 22 2 2 D-Mannitol 2222222 2 2 Salicin 222222+ 22 Sorbitol 22222+ 222 Rhamnose 2222222(+) 2 Maltose + 222(+) 2 (+)(+) 2 D-Xylose + 222(+) 2 + (+) 2 Trehalose + 222222 2 2 Cellobiose 22 2 2(+) 2 (+)(+) 2 Methyl D-glucoside 222222+ 22 Melibiose + 22222(+)(+) 2 D-Mannose + 22222(+)(+) 2 Hydrolysis of: Aesculin ++ + 2 + 2 (+)(+) + pNP-b-D-glucuronide 22(+) +++2 + 2 pNP-phosphoryl-choline ++ 2 +++2 + 2 L-Alanine-pNA +++++2 +++ L-Proline-pNA ++ + 2222 ++ Assimilation of: | D-Fructose ++++ 2 + 22+ Gluconate 22 + 2| 222 ++ a-Melibiose + 2 ++++2 + 2 L-Rhamnose 2222222(+) 2 Salicin + 2 ++++2 + 2 Maltitol 22 ++2 + 222 Acetate + 2 ++++2 + 2 Propionate 22 2 + 2 + 2 + 2 cis-Aconitate 22 + 222+++ trans-Aconitate 22 + 222+ 2 + Citrate ++ + 2222 + 2 Fumarate +++++++ 2 + Glutarate ++ + 22+ 2 + 2 DL-3-Hydroxybutyrate 2 +++++2 ++ DL-Lactate + 2 ++2 + 2 + 2 Oxoglutarate ++ + + 22+++ L-Alanine 2 +++++2 + (+) L-Asparate 22 2 + 2 + 222 L-Leucine 2 + 2 + 2 + 2 + (+) L-Ornithine 22 2 + 222 2 2 L-Proline 2 ++++22 2 + L-Serine 22222+ 222 4-Hydroxybenzoate 22222+ 222

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0.10 Sphingomonas zeae JM–791T (KP999966) Sphingomonas paucimobilis ATCC 29837T (U37337) 92 Sphingomonas sanguinis NBRC 13937T (D84529) Sphingomonas roseiflava MK341T (D84520) Sphingomonas yabuuchiae GTC 868T (AB071955) Sphingomonas parapaucimobilis NBRC 15100T (D13724) Sphingomonas pseudosanguinis G1−2T (AM412238) 100 Sphingomonas phyllosphaerae FA2T (AY453855) Sphingomonas endophytica YIM 65583T (HM629444) Sphingomonas yunnanensis YIM 003T (AY894691) Sphingomonas ginsenosidimutans Gsoil 1429T (HM204925) Sphingomonas polyaromaticivorans B2−7T (EF467848) 100 Sphingomonas oligoaromativorans SY-6T (FJ434127) Sphingomonas desiccabilis CP1DT (AJ871435) 73 Sphingomonas molluscorum An 18T (B248285) Sphingomonas pituitosa EDIVT (AJ243751) 94 Sphingomonas trueperi LMG 2142T (X97776) Sphingomonas azotifigens NBRC 15497T (AB03397) Sphingomonas guangdongensis 9NM−8T (JQ608326) Sphingomonas aerophila 5413J−26T (KC735148) 100 Sphingomonas ginsengisoli DCY58T (JN852951) Sphingomonas gei ZFGT−11T (KF551181) 79 Sphingomonas naasensis KIS18−15T (KC735149) Sphingomonas leidyi ATCC 15260T (AJ227812) Sphingomonas kyeonggiensis THG−DT81T (KC252615) Sphingomonas sanxanigenens NX02T (DQ789172) Stakelama sediminis CJ70T (EU099873) 88 Sphingomonas dokdonensis DS−4T (DQ178975) Sphingomonas mucosissima CP173−2T (AM229669) Sphingomonas xinjiangensis 10−1−84T (FJ754464) Sphingomonas japonica KC7T (AB428568) Sphingomonas yantingensis 1007T (JX566547) 100 Sphingomonas aestuarii K4T (EF660755) 'Sphingomonas hunanensis' JSM 083058T (FJ527417) T5−04T (AB166883) Sphingomonas pruni NBRC 15498T (Y09637) Sphingomonas mali NBRC 15500T (Y09638) Sphingomonas asaccharolytica NBRC 15499T (Y09639) JSS26T (AF131296) 86 T Sphingomonas panni C52 (AJ575818) Sphingomonas hankookensis ODN7T (FJ194436) Sphingomonas cynarae SPC−1T (HQ439186) Sphingomonas jinjuensis YC6723T (EU707561) Sphingomonas rubra BH3T (FJ834325) 77 Sphingomonas aquatilis JSS−7T (AF131295) PG−224T (AB055863) Sphingomonas kyungheensis THG-B283T (JN196137) Sphingomonas insulae DS−28 T (EF363714) Sphingomonas aerolata NW12T (AJ429240) 76 Sphingomonas aurantiaca MA101bT (AJ429236) 96 MA−olkiT (AJ429239) 'Sphingomonas ginsenosidivorax' KHI67 (HM204924) Sphingomonas abaci C42T (AJ575817) Sphingomonas echinoides ATCC 14820T (AB021370) Sphingomonas glacialis C16yT (GQ253122) Sphingomonas oligophenolica S213T (AB018439) Sphingomonas alpina S8−3T (GQ161989) Sphingomonas canadensis FWC47T (HE974351) 100 Sphingomonas jejuensis MS−31T (HQ224549) Sphingomonas gimensis 9PNM−6T (JQ608327) 76 Sphingomonas changbaiensis V2M44T (EU682685) Sphingomonas astaxanthinifaciens TDMA−17T (AB277583) 'Sphingomonas swuensis' PB62T (HQ844269) 'Sphingomonas rosea' PB196T (AB220121) Sphingomonas kaistensis PB56T (AY769083) Sphingomonas ginsengisoli Gsoil 634T (AB245347) Sphingomonas sediminicola KCTC 12629T (AB258386) 73 Sphingomonas daechungensis CH15−11T (JQ77248) Sphingomonas jaspsi TDMA−16T (AB264131) Sphingomonas oryziterrae YC6722T (EU707560) 92 Sandarakinorhabdus limnophila so42T (AY902680) Polymorphobacter multimanifer 262−7T (AB649056) 76 Sandaracinobacter sibiricus RB16-17T (Y10678)

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Fig. 1. Phylogenetic placement of strain JM-791T within the genus Sphingomonas. The analysis is based on 16S rRNA gene sequences (gene termini: 84 to 1392) and was generated in ARB (LTP database) with the maximum-parsimony methods using DNAPARS. Numbers at nodes represent bootstrap values based on 100 replications (bootstrap-analysis); only bootstrap values .70 % are displayed. Filled circles indicate nodes which were also present in the phylogenetic tree generated with the maximum-likelihood method; larger filled circles represent nodes which were thereby supported by bootstrap values .70 %. Type strains of species of the genera Sandaracinobacter and Polymorphobacter were used as outgroup. Bar, 0.1 substitutions per nucleotide position.

trehalose and melibiose, and hydrolyse many chromogenic analysed as described by Busse & Auling (1988) applying substrates. The biochemical/physiological data are given in HPLC conditions described by Busse et al. (1997). HPLC Table 1 and in the species description. apparatus used for analysis of quinones and polyamines was described by Stolz et al. (2007). For phylogenetic identification, the nearly full-length 16S rRNA gene of strain JM-791T was amplified and sequenced The detection of a quinone system consisting of ubiquinone using the universal primers 8F and 1492R (Lane, 1991). Q-10 (98 %), Q-11 (1 %) and Q-9 (1 %) in strain JM-791T After manual sequence correction, the sequence used for corresponded with the characteristics of species of the analysis had a length of 1401 nt spanning gene termini 15 genus Sphingomonas sensu stricto (Busse et al., 1999; to 1470 (according to Escherichia coli numbering; Brosius Kosako et al., 2000). The polyamine pattern exhibited the et al., 1978). The EzTaxon server (http://www.ezbiocloud. predominance of sym-homospermidine [41.6 mmol (g dry 2 net/eztaxon; Kim et al., 2012) was used to determine the weight) 1], the key characteristic of Sphingomonas sensu 16S rRNA gene sequence similarity to the closest related stricto (Busse et al., 1999; Takeuchi et al., 2001), and minor 2 type strains. Phylogenetic trees including type strains of all amounts of spermidine [1.7 mmol mmol (g dry weight) 1], 2 species of the genus Sphingomonas were calculated with the spermine [0.3 mmol (g dry weight) 1], and putrescine and 2 ARB software package release 5.2 (Ludwig et al., 2004) and cadaverine (eacg 0.1 mmol (g dry weight) 1]. The polar the ‘All-Species Living Tree’ Project (LTP; Yarza et al., lipid profile of strain JM-791T contained the major com- 2008) database release LTPs119 (November 2014). The pounds phosphatidylethanolamine, phosphatidylglycerol, T sequence of strain JM-791 was aligned with the SILVA Incre- diphosphatidylglycerol, sphingoglycolipid, phosphatidyl- mental Aligner (SINA; version 1.2.11; Pruesse et al., 2012) and choline, and a hydrophobic unidentified phospholipid implemented into the LTP database. The final alignment (PL1). In addition, moderate to minor amounts of one uni- used for tree reconstruction was checked manually consider- dentified aminolipid (AL1), one unidentified glycolipid ing the secondary structure information of the 16S rRNA. (GL1), another unidentified phospholipid (PL2), an uniden- A maximum-likelihood tree was generated on the basis of tified phosphoglycolipid (PGL1) and one lipid (L1) only sequences of the most closely related species using RAxML detectable after total lipid staining were also present (Fig. version 7.04 (Stamatakis, 2006) with GTR-GAMMA and 2). This profile shares the major characteristics of species rapid bootstrap analysis (100 resamplings) and a maxi- of Sphingomonas sensu stricto (Busse et al., 1999). mum-parsimony tree was created using DNAPARS version 3.6 (Felsenstein, 2005). Both trees were based on 16S rRNA Gas chromatography on the basis of the MIDI system was gene sequences between gene termini 84 to 1392 (according used to analyse the fatty acid profiles of the strains, as to Brosius et al., 1978) and based on 100 replications (boot- described previously (Ka¨mpfer & Kroppenstedt, 1996; T strap analysis; Felsenstein, 1985). Ka¨mpfer et al., 1997). Strain JM-791 showed a fatty acid profile typical for members of this genus with C14 : 0 2-OH Type strains of Sphingomonas paucimobilis (98.4 %), Sphin- as the key hydroxylated fatty acid (Table 2). gomonas pseudosanguinis (97.5 %) and Sphingomonas yabuu- T chiae (97.4 %) shared the highest 16S rRNA gene sequence Due to the relatively high similarity of strain JM-791 to similarities with strain JM-791T; the sequence similarity to type strains of S. paucimobilis, S. sanguinis and S. all other species of the genus Sphingomonas was below yabuuchiae, DNA–DNA hybridization experiments were 97.0 %. Both phylogenetic trees showed a clear placement performed with the type strains of these three species of strain JM-791T within the genus Sphingomonas, clustering using the method described by Ziemke et al. (1998), with the type strain of the type species S. paucimobilis, but not except that for nick translation, 2 mg DNA was labelled supported by high bootstrap values (Fig. 1). during a 3 h incubation at 15 8C. Strain JM-791T showed relatively low DNA–DNA relatedness to S. paucimobilis Chemotaxonomic analyses were performed as follows: res- DSM 1098T (51 %; reciprocal 37 %), S. pseudosanguinis piratory quinones and polar lipids were extracted from cells G1-2T (60 %; reciprocal 53 %) and S. yabuuchiae DSM harvested at the stationary growth phase determined 14562T (20 %; reciprocal 58 %). according to Tindall (1990a,b) and Altenburger et al. (1996), respectively; polyamines were extracted from cells On the basis of the 16S rRNA gene sequence analysis, harvested at the late exponential growth phase and DNA–DNA hybridization values and the phenotypic data,

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Table 2. Fatty acid composition of strain JM-791T and type L1 strains of selected species of the genus Sphingomonas DPG Strains: 1, JM-791T;2,S. pseudosanguinis G1-2T;3,S. sanguinis NBRC PL1 13937T;4,S. yabuuchiae A1-18T;5,S. paucimobilis ATCC 29837T;6, S. parapaucimobilis JCM 7510T;7,S. trueperi ATCC 12417T. Data were taken from the present study and Ka¨mpfer et al. (2007), AL1 PE obtained under exactly the same conditions. Values are percentages SGL of total fatty acids. ECL, Equivalent chain-length; TR, trace , 2 PG ( 1.0 %); , not detected.

Fatty acid 1 2 3 4 5 6 7 PC C14 : 0 2 1.0 1.0 1.3 1.4 1.0 2 C14 : 0 2-OH 12.4 5.4 6.7 8.7 6.4 5.0 6.7 C15 : 0 2-OH 1.5 222222 C 222221.1 2 GL1 15 : 0 C 13.3 12.9 9.6 9.9 8.7 13.6 9.8 PL2 16 : 0 C16 : 1v5c 221.9 TR 2 1.0 2 2 2 PGL1 Summed feature 4* 4.8 8.6 5.9 2.7 6.7 C17 : 0 1.6 221.2 2 0.5 1.6 st nd 1 dimension 2 dimension C17 : 1v6c 4.7 0.8 3.8 5.1 3.0 2.4 13.6 C18 : 0 1.3 1.6 2 TR 2 0.6 2 T Fig. 2. Polar lipid profile of JM-791 after two-dimensional thin Summed feature 7* 63.8 72.0 65 62.5 74.6 64.6 64.2 layer chromatography and detection with molybdatophosphoric C18 : 1v5c 1.4 1.4 3.5 2.3 3.2 3.5 4.0 acid. DPG, diphosphatidylglycerol; PG, phosphatidylglycerol; PE, phosphatidylethanolamine; PC, phosphatidylcholine; SGL, sphin- *Summed features represent groups of two or three fatty acids that goglycolipid; PL1, 2, unidentified phospholipids; AL1, unidentified could not be separated by GLC with the MIDI system (Microbial aminolipid; PGL1, unidentified phosphoglycolipid; GL1, unidenti- ID). Summed feature 4 contained one or more of C16 : 1v7t, fied glycolipid; L1, unidentified polar lipid. C15 : 0 iso 2-OH and C16 : 1v7c; summed feature 7 contained one or more of C18 : 1v7c,C18 : 1v9t and/or C18 : 1v12t. strain JM-791T represents a novel species of the genus Sphingomonas, for which we propose the name Sphingomo- phenyl phosphonate, 2-deoxythymidine-59-pNP-phosphate, nas zeae sp. nov. pNP-phosphoryl choline, L-alanine-pNA, L-glutamate-c- 3-carboxy-pNA and L-proline-pNA, but not pNP-b-D- Description of Sphingomonas zeae sp. nov. glucuronide. The species is characterized by the major fatty acids C ,C v7c,C v9t and/or C v12t Sphingomonas zeae [L. gen. n. zeae of spelt, of Zea mays, 18 : 1 18 : 1 18 : 1 18 : 1 (detected as summed feature 7) and C and the major referring to its isolation from the stem of corn (Zea mays)]. 16 : 0 hydroxy-fatty acid C14 : 0 2-OH. The major polyamine is Cells are rod-shaped, 0.8–1.5 mm in length and 0.4–0.6 mm sym-homospermidine; minor polyamines are putrescine, in diameter. Yellow colonies are formed. Acid is produced cadaverine, spermidine and spermine. The quinone from D-glucose, sucrose, lactose (weak), maltose, D-xylose, system is ubiquinone Q-10 with minor amounts of trehalose and melibiose, but not from D-mannitol, salicin, Q-9 and Q-11. The polar lipid profile is composed dulcitol, adonitol, i-inositol, D-sorbitol, L-rhamnose, cello- of the major lipids phosphatidylethanolamine, phosphati- biose, erythritol or D-arabitol. Uses N-acetyl-D-glucosa- dylglycerol, diphosphatidylglycerol, sphingoglycolipid, mine, L-arabinose, p-arbutin, cellobiose, D-fructose, D- phosphatidylcholine, and an unidentified phospholipid galactose, D-glucose, D-mannose, maltose, melibiose, (PL1). In addition, moderate to minor amounts of an uni- sucrose, salicin, trehalose, D-xylose, acetate, citrate, fuma- dentified aminolipid (AL1), one unidentified glycolipid rate, glutarate, DL-lactate and L-malate as substrate, (GL1), another unidentified phospholipid (PL2), an uni- but not gluconate, L-rhamnose, ribose, D-adonitol, maltitol, dentified phosphoglycolipid (PGL1) and one lipid (L1) D-mannitol, D-sorbitol, propionate, DL-3-hydroxybutyrate, are present. azelate, cis-aconitate, adipate, 4-aminobutyrate, pyruvate, T T T L-alanine, L-leucine, L-aspartate, L-histidine, L-ornithine, L- The type strain JM-791 (5LMG 28739 5CCM 8596 ), phenylalanine, L-proline, L-tryptophan, 3-hydroxybenzoate, was isolated from the healthy internal stem tissue of 1- 4-hydroxybenzoate or phenylacetate. Hydrolysesaesculin, month-old corn (Zea mays, cultivar ‘Sweet Belle’) grown pNP-a-D-glucopyranoside, pNP-b-D-glucopyranoside, at the Plant Breeding Unit of the E.V. Smith Research pNP-b-D-galactopyranoside, bis-pNP-phosphate, pNP- Center in Tallassee (Elmore county), Alabama, USA.

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