Microbacterium Shaanxiense Sp. Nov., Isolated from the Nodule Surface of Soybean

International Journal of Systematic and Evolutionary Microbiology (2015), 65, 1437–1443 DOI 10.1099/ijs.0.000116

Microbacterium shaanxiense sp. nov., isolated from the nodule surface of soybean

Shi Peng, Liu Dongying, Yang Bingxin, Li Mingjun and Wei Gehong

Correspondence State Key Laboratory of Soil Erosion and Dryland Farming on the Loess Plateau, College of Life Wei Gehong Sciences, Northwest A&F University, Yangling, Shaanxi 712100, PR China [email protected]

A Gram-stain-positive, non-motile, catalase- and oxidase-positive rod, designated CCNWSP60T, was isolated from the nodule surface of soybean [Glycine max (L.) Merrill] cultivar Zhonghuang 13. 16S rRNA gene sequence analysis clearly showed that the isolate belonged to the genus Microbacterium. On the basis of pairwise comparisons of 16S rRNA gene sequences, strain CCNWSP60T was most closely related to Microbacterium murale DSM 22178T (98.8 % similarity), Microbacterium aerolatum DSM 14217T (98.3 %), Microbacterium ginsengiterrae DSM 24823T (98.0 %) and Microbacterium profundi DSM 22239T (97.8 %). However, the DNA– DNA relatedness values of strain CCNWSP60T to M. murale DSM 22178T, M. aerolatum DSM 14217T, M. ginsengiterrae DSM 24823T and M. profundi DSM 22239T were 48 %, 43 %, 28 % and 41 %, respectively. Growth of strain CCNWSP60T occurred at 4–40 6C and at pH 5.0–9.0. The NaCl range for growth was 0–4 % (w/v). The predominant menaquinone of strain CCNWSP60T was MK-13; MK-12 was also detected. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, one unidentified glycolipid and one unidentified phospholipid. The diagnostic diamino acid of the peptidoglycan was ornithine. The acyl type of the

peptidoglycan was glycolyl. The major fatty acids were anteiso-C15 : 0, iso-C16 : 0 and C16 : 0. The DNA G+C content of the type strain was 67.4 mol%. As the physiological and biochemical characteristics as well as the DNA–DNA relatedness between strain CCNWSP60T and the type strains of its closest phylogenetic neighbours showed clear differences, a novel species Microbacterium shaanxiense is proposed to accommodate it. The type strain is CCNWSP60T (5DSM 28301T5ACCC 19329T5JCM 30164T).

The genus Microbacterium (phylum Actinobacteria,classActino- rhizosphere soil was removed from the roots by shaking bacteria,orderActinomycetales, family Microbacteriaceae) was vigorously, and then all nodules were picked off using aseptic proposed by Orla-Jensen (1919) and was emended by forceps and washed with sterile water (363 min) to remove Collins et al. (1983). Then, the genus was emended by bacterial cells that did not adhere to the nodule surface. The Takeuchi & Hatano (1998) to unite the closely related genera nodules were then placed in an Erlenmeyer flask containing Microbacterium and Aureobacterium. At the time of writing, glass beads and saline solution [0.7 % (w/v) NaCl]. The flask the genus Microbacterium comprises 82 species with validly was shaken at 150 r.p.m., 28 uC for 1 h. After agitation, published names (www.bacterio.net). Members of this genus appropriate dilutions of the contents in the flasks were plated are widely distributed and have been isolated from various onto Luria–Bertani (LB) agar and incubated at 28 uC. habitats, such as soil (Kim et al., 2010), walls (Ka¨mpfer et al., The strain was obtained by repeated transfers of single 2012) and deep-sea sediment (Wu et al., 2008). The novel colonies on tryptic soy agar, which contained 15.0 g agar strain we describe here was isolated from the surface of in 1 L tryptic soy broth at 28 uC. A polyphasic approach, soybean nodule. including physiological, chemotaxonomic and phylogenetic Strain CCNWSP60T was isolated from the nodule surface analyses, was used to determine the taxonomic position and of soybean [Glycine max (L.) Merrill] cultivar Zhonghuang identity of this novel strain. The four related reference 13 from a field in Yangling, Shaanxi province of PR China strains used in our study were Microbacterium murale DSM T T (34u 169 N 108u 49 E). To isolate nodule epiphytic bacteria, 22178 , Microbacterium aerolatum DSM 14217 , Microbac- terium ginsengiterrae DSM 24823T and Microbacterium profundi DSM 22239T (from Deutsche Sammlung von The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene Mikroorganismen und Zellkulturen, DSMZ). sequence of strain CCNWSP60T is KJ735510. T Three supplementary figures are available with the online Supple- Strain CCNWSP60 was cultivated on tryptic soy agar at mentary Material. 28 uC for 72 h for morphological investigations of colonies.

000116 G 2015 IUMS Printed in Great Britain 1437 S. Peng and others

Cell morphology and Gram staining were observed by light DNA–DNA hybridizations were performed with M. murale microscopy (CX31; Olympus) after 72 h of incubation on DSM 22178T, M. aerolatum DSM 14217T, M. ginsengiterrae tryptic soy agar at 28 uC. Scanning electron microscopy (S- DSM 24823T and M. profundi DSM 22239T by the thermal 4800; Hitachi) was also used to observe cell morphology. denaturation and renaturation method of De Ley et al. Cell motility was determined using the hanging drop method (1970) using a PerkinElmer Lambda 35 UV/Vis Spectrom- (Bernardet et al., 2002). Strain CCNWSP60T formed circular, eter. The DNA G+C content of strain CCNWSP60T was smooth, pale-yellow, slightly convex colonies with entire 67.4 mol%, consistent with the DNA G+C contents of margins, which were approximately 0.5–1 mm in diameter members of the genus Microbacterium (Takeuchi & after 72 h of incubation. Cells of strain CCNWSP60T were Hatano, 1998). The DNA relatedness values of strain Gram-stain-positive, non-motile and rod-shaped (0.3– CCNWSP60T to M. murale DSM 22178T, M. aerolatum 0.4 mminwidth60.8–1.3 mm in length) (Fig. S1, available DSM 14217T, M. ginsengiterrae DSM 24823T and M. in the online Supplementary Material). profundi DSM 22239T were 48 %, 43 %, 28 % and 41 %, respectively. The genomic DNA was extracted using the method of Zhao et al. (2010) and the 16S rRNA gene was amplified by PCR Catalase activity was determined by production of bubbles with primers 8F (59-AGAGTTTGATC MTGGCTCAG-39) after mixing cells from colonies grown on tryptic soy agar and 1492 R (59-TACGG(C/T)TACCTTGTTACGACTT-39) with 3 % (v/v) H2O2. Oxidase activity was tested by using (Blackwood et al., 2007) and sequenced using the method API oxidase reagent according to the manufacturer’s instruc- of Sanger dideoxy sequencing with the same pair of primers. tions (bioMe´rieux). Hydrolysis of casein, starch, and Tweens The almost-complete 16S rRNA gene sequence (1425 nt) of 20 and 80 were tested according to methods of Smibert & strain CCNWSP60T was compared to publicly available Krieg (1994). Carbon source utilization was tested using a sequences of related type strains, and 16S rRNA gene Biolog GP2 MicroPlate according to the manufacturer’s sequence similarities were calculated using the EzTaxon-e instructions. Enzyme activities were analysed using the API server (http://eztaxon-e.ezbiocloud.net/; Kim et al., 2012). ZYM kit (bioMe´rieux), acid production from carbohydrates The results showed that the closest relatives were members and other physiological and biochemical properties were of the genus Microbacterium. Strain CCNWSP60T showed analysed using the API 50 CH and API 20NE systems 98.8 % similarity to M. murale DSM 22178T, 98.3 % to M. (bioMe´rieux), according to the manufacturer’s instructions. aerolatum DSM 14217T, 98.0 % to M. ginsengiterrae DSM Further phenotypic properties of strain CCNWSP60T are 24823T, 97.8 % to M. profundi DSM 22239T and less given in Table 1 and in the species description. than 97.6 % similarity to other members of the genus The menaquinones of strain CCNWSP60T were extracted Microbacterium. Multiple alignments were carried out using according to the method of Collins et al. (1977) and analysed CLUSTAL X (Thompson et al., 1997). Phylogenetic trees were using reversed-phase HPLC (Groth et al., 1996), with inferred by using the neighbour-joining (Saitou & Nei, menaquinones from M. murale DSM 22178T (MK-10, 11, 12 1987) and maximum-likelihood (Felsenstein, 1981) algo- and 13) and M. profundi DSM 22239T (MK-12 and 13) as rithms with the MEGA 5.2. program. Evolutionary distances reference standards. Polar lipids were extracted as described were calculated using Kimura’s two-parameter model. The by Minnikin et al. (1984) and identified by two-dimensional topology of the phylogenetic tree was evaluated by the TLC and spraying with specific reagents as described by bootstrap resampling method of Felsenstein (1985) with Collins & Jones (1980). Acid-fast staining was carried out 1000 replicates. The neighbour-joining phylogenetic tree using the Kinyoun Solution (BKK-F)–Gabett Solution showed that strain CCNWSP60T formed an intragenus branch (BKG-F) (bioMe´rieux) according to the manufacturer’s with M. ginsengiterrae DSM 24823T (Fig. 1). The maximum- instructions. The predominant menaquinone of strain likelihood tree showed the same topology (Fig. S2). CCNWSP60T was MK-13 (about 76 %), and some MK-12 To examine the optimal conditions for growth, strains, (about 24 %) was also detected. The major polar lipids were including CCNWSP60T and some strains of closely related diphosphatidylglycerol, phosphatidylglycerol, one unknown species, were cultured in tryptic soy broth (BEIJING LAND glycolipid and one unknown phospholipid (Fig. S3). The BRIDGE TECHNOLOGY CO., LTD., China) medium at major menaquinones and polar lipids were similar to those different temperatures (4, 10, 20, 25, 28, 30, 37, 40, 45 and of some members of the genus Microbacterium as described 50 uC), pH values (4, 5, 6, 7, 8, 9, 10, 11) and salt by Takeuchi & Hatano (1998). The strain was not acid-fast. concentrations [0, 1, 2, 3, 4, 5, 6, 7, 8 and 9 % NaCl (w/v)]. For quantitative analysis of fatty acids, strains were All tests were carried out by incubating cultures at 28 uC cultivated on tryptic soy agar at 28 uC for 72 h. The and pH 7, except for investigations of the effect of preparation and analysis of fatty acids were conducted temperature and pH. Strain CCNWSP60T grew at 4– according to the standard protocol of the MIDI Sherlock 40 uC (optimum, 28 uC), pH 5–9 (optimum, pH 7) and Microbial Identification System (Microbial ID) (Khan et al., NaCl concentrations of 0–6 % (w/v). 2008). The major fatty acids (.10 %) of strain CCNWSP60T The DNA G+C content was determined by using reversed- were anteiso-C15 : 0 (48.2 %), iso-C16 : 0 (20.4 %) and C16 : 0 phase HPLC according to Mesbah et al. (1989), using (12.1 %). The fatty acid composition of strain CCNWSP60T Escherichia coli K-12 DNA as the calibration standard. was different from those of the reference strains (Table 2).

1438 International Journal of Systematic and Evolutionary Microbiology 65 Microbacterium shaanxiense sp. nov.

96 M. maritypicum DSM 12512T (AJ853910) M. oxydans DSM 20578T (Y17227) 84 M. luteolum NBRC 15074T (AB004718) 0.005 M. saperdae NBRC 15038T (AB004719) M. paraoxydans CF36T (AJ491806) M. testaceum DSM 20166T (X77445) M. resistens DMMZ 1710T (Y14699) M. azadirachtae AI-S262T (EU912487) M. esteraromaticum DSM 8609T (Y17231) 98 M. arabinogalactanolyticum NBRC 14344T (AB004715) M. profundi Shh49T (EF623999) M. shaanxiense CCNWSP60T (KJ735510) 72 M. ginsengiterrae DCY37T (EU873314) M. oleivorans DSM 16091T (AJ698725) M. keratanolyticum NBRC 13309T (AB004717) M. natoriense TNJL143-2T (AY566291) M. aerolatum V-73T (AJ309929) M. phyllosphaerae DSM 13468T (AJ277840) 87 M. foliorum DSM 12966T (AJ249780) M. arthrosphaerae CC-VM-YT (FN870023) M. hydrocarbonoxydans DSM 16089T (AJ698726) M. murale 1-Gi-001T (HE585693) 100 M. terrae NBRC 15300T (AB004720) M. ketosireducens NBRC 14548T (AB004724) M. aurantiacum DSM 12506T (AM182159) Kitami C2T (AB013919) 100 M. kitamiense M. aurantiacum CIP 105730T (EU863415) M. binotii CIP 101303T (EF567306) M. lemovicicum VKM Ac-1782 (AB042073) 100 M. lemovicicum ATCC BAA-2396T (JN243353) M. pumilum KV-488T (AB234027) 98 M. lacus A5E-52T (AB286030) Gaciihabitans tibetensis MP203T (KC256953)

Fig. 1. Neighbour-joining tree based on 16S rRNA gene sequences of strain CCNWSP60T and some type strains of species of the genus Microbacterium. Glaciihabitans tibetensis MP203T was used as an outgroup. Bootstrap values (expressed as percentages of 1000 replications) .70 % are shown at nodes. Bar, 0.005 substitutions per nucleotide position.

To analyse amino acids and sugars, the cells of strain 1980). The acyl type of the peptidoglycan was tested by the CCNWSP60T were hydrolysed in 6 M HCl at 100 uC for method of Uchida et al. (1999). The peptidoglycan 16 h and in 0.5 M HCl at 100 uC for 2 h, respectively. Then hydrolysates of CCNWSP60T contained glycine, alanine, the hydrolysates were analysed by TLC on microcrystalline glutamic acid and ornithine, which could also be detected cellulose plates ( KGaA; Merck) and visualized by spraying in the reference strain M. murale DSM 22178T, indicating ninhydrin reagent for detection of amino acids and aniline that the diagnostic diamino acid of the peptidoglycan was acid phthalate reagent for detection of sugars (Becker et al., ornithine. The acyl type of the peptidoglycan was glycolyl. 1965; Staneck & Roberts, 1974; Lechevalier & Lechevalier, The whole-cell sugars were xylose, ribose and galactose. http://ijs.sgmjournals.org 1439 S. Peng and others

Table 1. Differential characteristics of strain CCNWSP60T and closely related species

Strains: 1, CCNWSP60T;2,M. murale DSM 22178T;3,M. profundi DSM 22239T;4,M. aerolatum DSM 14217T;5,M. ginsengiterrae DSM 24823T. All data from the present study. +, Positive; 2, negative.

Characteristic 1 2 3 4 5

Growth temperature (uC) 4–40 4–37 10–37 4–40 4–40 NaCl range for growth (%, w/v) 0–6 0–7 0–7 0–7 0–7 pH range for growth 5–9 6–9 5–9 6–8 5–8 Reduction of nitrates to nitrites 2 + 2 + 2 Hydrolysis of: Aesculin ferric citrate ++222 Gelatin 2222+ Tween 20 +++2 + Utilization of (Biolog GP2 microplate): a-Cyclodextrin 22+ 2 + Glycogen 22+ 22 Mannan 22++2 Tween 40 +++2 + N-Acetyl-D-galactosamine 22+++ Amygdalin 2 + 2 ++ Arbutin ++22+ D-Galacturonic acid 222++ Gentiobiose +++2 + myo-Inositol 2222+ a-Lactose 22+++ Lactulose 22+++ Methyl a-D-galactoside 222++ Methyl b-D-galactoside 22+++ Methyl b-D-glucoside 22+ 2 + Methyl a-D-mannoside + 2 +++ Sedoheptulosan 222++ Stachyose 2222+ Xylitol +++22 Acetic acid +++22 b-Hydroxybutyric acid 2 + 2 ++ c-Hydroxybutyric acid 222++ a-Ketoglutaric acid 2222+ a-Ketovaleric acid ++++2 Lactamide 22+++ Succinamic acid + 2 +++ Succinic acid + 2 +++ N-Acetyl-L-lactamide glutamic acid + 2 +++ L-Alaninamide ++++2 L-Alanyl-glycine +++2 + L-Asparagine + 2 +++ L-Glutamic acid ++++2 Glycyl-L-glutamic acid 22+++ Putrescine +++2 + 2,3-Butanediol 222+ 2 Glycerol ++++2 Adenosine ++222 29-Deoxyadenosine ++222 Inosine ++222 Thymidine ++222 Uridine ++222 Adenosine-59-monophosphate ++222 Thymidine-59-monophosphate ++222 Uridine-59-monophosphate 2 + 222

1440 International Journal of Systematic and Evolutionary Microbiology 65 Microbacterium shaanxiense sp. nov.

Table 1. cont.

Characteristic 1 2 3 4 5

DL-a-Glycerol phosphate + 2222 Acid production from (API 50CH): Glycerol +++2 + Erythritol ++++2 Adonitol ++222 Methyl b-D-xylopyranoside 22+ 22 D-Sorbitol + 2222 Methyl a-D-mannopyranoside ++222 Methyl a-D-glucopyranoside ++++2 Amygdalin 2 + 2 ++ Arbutin ++22+ Aesculin ++222 Salicin ++22+ Glycogen 22+ 22 Xylitol +++22 Gentiobiose +++2 + D-Arabitol + 2222 Potassium gluconate ++2 ++ DNA G+C content (mol%) 67.4* 66.0* 65.9* 68.0* 68.6*

*Mean value from two tests.

Description of Microbacterium shaanxiense ranges for growth are 4–40 uC, pH 5–9 and 0–6 % (w/v) sp. nov. NaCl, respectively. Catalase and oxidase are present. Casein, starch, and Tweens 20 and 80 are hydrolysed. In Microbacterium shaanxiense (shaan.xi.en9se. N.L. neut. adj. the API 20NE system, nitrate is not reduced to nitrite or shaanxiense pertaining to Shaanxi, the locale where the type nitrogen. Indole production, D-glucose fermentation, L- strain was isolated). arginine dihydrolase, urease and gelatin hydrolysis are Cells are rod-shaped, non-motile, Gram-stain-positive, negative. Aesculin ferric citrate hydrolysis and b-galacto- 0.3–0.4 mm in width and 0.8–1.3 mm in length. Colonies sidase tests are positive. D-Glucose, L-arabinose, D-man- are pale-yellow, smooth, circular, 0.5–1 mm in diameter nose, D-mannitol, maltose, potassium gluconate and malic and slightly convex with entire margins after growth on acid can be assimilated, but N-acetylglucosamine, capric tryptic soy agar for 72 h. The temperature, pH and NaCl acid, adipic acid, trisodium citrate and phenylacetic acid cannot. In the API ZYM system, positive for esterase C4, leucine arylamidase, valine arylamidase, acid phosphatase, b a T naphthol-AS-BI-phosphohydrolase, -galactosidase, -glu- Table 2. Cellular fatty acid contents of strain CCNWSP60 cosidase, b-glucosidase, N-acetyl-b-glucosaminidase and and related members of the genus Microbacterium a-mannosidase, but negative for alkaline phosphatase, Strains: 1, CCNWSP60T;2,M. murale DSM 22178T;3,M. profundi esterase lipase C8, lipase C14, cystine arylamidase, trypsin, DSM 22239T;4,M. aerolatum DSM 14217T;5,M. ginsengiterrae DSM a-chymotrypsin, a-galactosidase, b-glucuronidase and a- 24823T. All data from the present study. 2, Not detected or ,1%. fucosidase. In the API 50 CH system, acid is produced from methyl a-D-glucopyranoside, methyl a-D-mannopyrano- Fatty acid (%) 1 2 3 4 5 side, D-arabinose, D-arabitol, D-mannitol, D-sorbitol, turanose, D-xylose, L-arabinose, L-fucose, adonitol, aesculin, iso-C14 : 0 3.35 3.21 1.73 1.48 2.03 arbutin, cellobiose, erythritol, fructose, galactose, gentio- iso-C 5.36 7.77 15.23 2.7 2.46 15 : 0 biose, glucose, glycerol, maltose, mannose, melezitose, anteiso-C15 : 0 48.18 48.56 50.82 51.71 53.89 iso-C 20.36 22.87 14.99 16.87 15.27 potassium 5-ketogluconate, potassium gluconate, rham- 16 : 0 nose, ribose, salicin, starch, sucrose, trehalose and xylitol, iso-C17 : 0 221.67 22 but not from methyl b-D-xylopyranoside, D-fucose, D- anteiso-C17 : 0 8.08 11.22 10.13 17.89 17.18 D L L C14 : 0 2.53 2 1.14 22 lyxose, -tagatose, -arabitol, -xylose, N-acetylglucosamine, C16 : 0 12.13 6.37 4.29 9.37 7.53 amygdalin, dulcitol, glycogen, inositol, inulin, lactose, anteiso-C15 : 1 A 22221.64 melibiose, potassium 2-ketogluconate, raffinose or sorbose. 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1442 International Journal of Systematic and Evolutionary Microbiology 65 Microbacterium shaanxiense sp. nov.

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