Antonie van Leeuwenhoek DOI 10.1007/s10482-015-0481-8
ORIGINAL PAPER
Mesorhizobium soli sp. nov., a novel species isolated from the rhizosphere of Robinia pseudoacacia L. in South Korea by using a modified culture method Tuan Manh Nguyen . Van Hong Thi Pham . Jaisoo Kim
Received: 19 March 2015 / Accepted: 13 May 2015 Ó Springer International Publishing Switzerland 2015
Abstract Strain NHI-8T was isolated from a forest soil sample, collected in South Korea, by using a modified culture method. Comparative analysis of its nearly full-length 16S rRNA gene sequence showed that strain NHI-8T belongs to the genus Mesorhizobium and to be closely related to Mesorhizobium chacoense PR5T (97.32 %). The levels of DNA– DNA relatedness between strain NHI-8T and reference type strains of the genus Mesorhizobium were 32.28–53.65 %. SDS-PAGE of total soluble proteins and the sequences of the housekeeping genes recA, glnII, and atpD were also used to support the clade grouping in rhizobia. The new strain contained summed feature 8 (57.0 %), cyclo-C19:0x8c (17.3 %), and C18:0 (11.0 %) as the major fatty acids, as in genus Mesorhizobium. The strain contained cardiolipin, phosphatidylglycerol, ornithinecontaining lipid, phosphatidylethanolamine,
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA, recA, atpD, and glnII genes are KC484966, KM188061, KM188059, and KM188060, respectively.
Electronic supplementary material The online version of this article (doi:10.1007/s10482-015-0481-8) contains supplementary material, which is available to authorized users. T. M. Nguyen V. H. T. Pham J. Kim (&) Department of Life Science, College of Natural Sciences, Kyonggi University, Suwon, Gyeonggi-Do 443-760, Republic of Korea e-mail: [email protected]
phosphatidyl-N-dimethylethanolamine, and phosphatidylcholine. Morphological and physiological analyses were performed to compare the characteristics of our strain with those of the reference type strains. Based on the results, strain NHI-8T was determined to represent a novel member of the genus Mesorhizobium, and the name Mesorhizobium soli is proposed. The type strain is NHI-8T (=KEMB 9005-153T = KACC 17916T = JCM 19897T). Keywords Mesorhizobium sp. nov. Housekeeping gene Taxonomy Modified culture method Rhizosphere Robinia pseudoacacia L
Introduction Bacteria are a large domain of prokaryotic microorganisms that are involved in various metabolic cycles, thus creating balance in nature. Within the nitrogen cycle, bacteria, in symbiotic association with plants, fix nitrogen, which helps to enhance host photosynthesis (Bethlenfalvay et al. 1978). One group of nitrogen-fixing bacteria, the rhizobia, are Gramnegative soil bacteria that are found in nodules on the roots of plants and fix nitrogen from the atmosphere into ammonia (Weir et al. 2004; Terpolilli et al. 2012). The host range for rhizobial species is limited, with approximately 200 known plant species that are mainly legumes (Weir et al. 2004). With the help of
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rhizobial inoculants, the yields of various plants have been increased, including soybeans in the United States (Lohrke et al. 1996), European Lotus corniculatus in New Zealand (Sullivan et al. 1995), and Asian Cicer arietinum (chickpea) in Australia (Howieson et al. 2000). The genus Mesorhizobium contains many distinctive characteristics from the genus Rhizobium. Jarvis et al. (1997) re-classified and re-described the genus Mesorhizobium with major characterisations; namely, the species are Gram-negative, aerobic, nonspore-forming, rod-shaped, and motile. They often form nitrogen-fixing nodules on the roots of a limited range of legume plants (Laranjo et al. 2014). Currently, there are 30 known species (http://www.bacterio. net/mesorhizobium.html), most of which were found in China. Strain NHI-8T was isolated from the rhizosphere of Robinia pseudoacacia in South Korea by using a modified culture method. In total, 12 species were found to be closely related to strain NHI-8T, the majority of which were isolated from the legume family (Table S1). To determine the taxonomic classification of our strain, we performed DNA–DNA hybridization with related species, determined its fatty acid profile and the sequences of its housekeeping genes (recA, atpD, and glnII), and collected other information on its morphology and physiology.
Materials and methods Isolation of strain Samples were collected from the surface soil covering plant roots in the forest at Kyonggi University in Suwon, Gyeonggi-do, South Korea. Bacteria were isolated by using a modified culture method, as described by Pham and Kim (2014). Briefly, 3 g of soil was added to each well of a transwell plate, and then 3 mL of R2A medium [(g L-1); proteose peptone, 0.5; yeast extract,0.5; acid digest of casein, 0.5; glucose, 0.5; soluble starch, 0.5; dipotassium phosphate, 0.3; magnesium sulphate, 0.024; and sodium pyruvate, 0.3; pH 7.2] was added into each transwell insert. Then, 100 lL of a soil suspension (containing 1 g of soil in 10 mL of R2A medium, thoroughly stirred and allowed to settle) was inoculated in each insert. The plate was incubated in a shaking incubator at 120 rpm and 28 °C for 2 weeks. After the
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2 weeks of incubation, serial dilutions of the culture were prepared. An aliquot (100 lL) of each dilution was spread on an R2A agar plate, and the plate was incubated at 28 °C for 5 days. Different types of colonies were picked and streaked on individual agar plates, and this was repeated until a pure colony was obtained. The pure colony was preserved in a 20 % glycerol stock at -20 °C. Phenotypic characterisation Various morphological and physiological tests were performed to compare the characteristics of our strain with those of type strains. The cell size was measured by microscopy (BX50 microscope; Olympus, Japan). Motility was assessed in tryptic soy broth medium (CM0129; Oxoid) containing 0.4 % agar. Gram staining was carried out according to the method of Doetsch (1981). Catalase activity was assessed by bubble production in 3 % (v/v) H2O2, and oxidase activity was determined by using 1 % (w/v) tetramethyl-p-phenylenediamine. Hydrolysis of starch, casein, and gelatin, hydrogen sulphide (H2S) production, and methyl red and Voges–Proskauer tests were determined as described by Smibert and Krieg (1994). Growth at different temperatures (4, 10, 15, 20, 25, 28, 30, 37, 40, 50 and 55 °C) and at various pH values (pH 4.0–12.0; at intervals of 0.5 pH unit) was determined with different buffers at 100 mM concentration (acetate buffer for pH 4–5; phosphate buffer for pH 5–8; Tris buffer for pH 9–12), and tolerance to various concentrations of NaCl (0–10 %, at 0.5 % intervals) was assessed after incubation for up to 5 days. Yeast mannitol broth medium [YMB (g L-1): manitol, 10; KH2PO4, 0.5; sodium glutamate, 0.5; NaCl, 50 9 10-3; solution A, 10 mL; solution B, 1 mL; solution C, 1 mL; yeast extract, 1; pH 6.8 (solution A: 1 g of MgSO47H2O in 100 mL of distilled water; solution B, 5.28 g of CaCl2H2O in 100 mL of distilled water; solution C, 666 9 10-3 g of FeCl36H2O in 100 mL of distilled water)] was used for determining the growth of each strain under different temperature and pH values, NaCl concentrations, and metal ion types. API 50CH (with API 50 CHB/E medium), ID 32GN, API 20NE, and API ZYM strips (bioMe´rieux, France) were used to compared phenotypic characteristics between the strain and type species of the genus Mesorhizobium (Table 1).
Antonie van Leeuwenhoek Table 1 Differential phenotypic characteristics between strain NHI-8T and closely related species of the genus Mesorhizobium Characteristic
1
2
3
4
5
6
7
8
9
10
11
12
13
?
-
-
?
?
-
-
-
?
-
-
w
?
? ?
? w
? -
? ?
? -
-
? -
w
?
? ?
? -
? -
-
Urea
?
-
-
-
-
-
?
-
-
-
?
?
-
Trisodium citratec
?
-
-
-
-
-
-
-
-
-
-
-
-
Esculin ferric citrateb
?
-
-
-
-
-
-
-
-
-
?
-
-
D-Fucose
?
?
-
-
-
-
-
-
?
-
?
-
-
?
-
?
-
-
?
?
-
w
-
?
-
-
?
?
-
?
?
-
?
?
?
-
?
?
-
D-Arabinose
?
-
?
-
?
-
?
?
?
?
w
?
-
D-Ribose
-
?
-
?
?
?
?
-
-
?
?
-
?
L-Xylose
-
-
-
-
-
-
w
-
-
-
?
-
?
D-Galactose
w
?
?
?
?
-
-
?
-
?
-
-
?
D-Fructose
w
-
?
?
?
-
-
?
w
-
-
?
?
D-Mannose
w
-
-
?
-
-
-
?
?
-
-
-
?
Assimilation of API 20NE D-Glucose
a
L-Arabinose D-Maltose
a
a
Acid from API 50CH
L-Fucose
c
D-Saccharose
c
L-Rhamnose
-
-
?
-
?
-
?
-
?
-
?
?
?
D-Sorbitol
w
-
-
? -
? -
-
-
-
? -
?
? ?
-
?
D-Trehalose
Xylitol
?
-
-
-
-
-
-
-
?
-
?
-
D-Xylose
?
-
-
?
?
w
-
?
?
-
-
?
?
D-Tagatose
-
-
-
-
-
-
-
-
?
?
-
-
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Inositol
-
-
-
-
-
?
w
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?
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?
?
Potassium gluconate
-
-
-
w
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?
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?
-
-
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?
Assimilation of ID 32GN Sodium acetate
?
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?
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?
-
-
-
-
-
Lactic acid
?
-
-
-
-
-
?
-
-
-
-
-
–
L-Alanine
?
-
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-
-
-
-
-
-
-
-
-
L-Proline
?
-
-
?
-
?
?
?
w
-
-
-
?
N-Acetyl-glucosamine
-
-
-
-
-
-
?
-
?
?
-
-
-
Enzymatic activities (API ZYM) Alkaline phosphatase
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?
?
?
?
?
w
?
w
?
?
?
?
Esterase (C4) Trypsin
w ?
w ?
? ?
? ?
? ?
? ?
? -
? ?
? ?
w ?
w ?
? w
w ?
a-Glucosidase
?
w
-
-
-
-
-
-
-
-
-
-
-
Ampicillin (0.5)
-
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?
?
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?
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?
?
?
?
-
?
Neomycin (5)
-
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?
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?
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?
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-
?
?
Rifampicin (0.25)
-
-
?
?
-
?
-
?
?
?
?
?
-
Zn2? (0.1)
?
?
?
?
-
?
?
?
?
?
?
?
?
Zn2? (0.3)
?
?
?
?
-
-
?
?
?
?
?
-
?
Zn2? (0.5)
?
?
?
?
-
-
?
-
-
?
-
-
?
Resistant to (lg mL-1)
Tolerance of (mM)
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Antonie van Leeuwenhoek Table 1 continued Characteristic
1
2
3
4
5
6
7
8
9
10
11
12
13
Zn2? (1) Zn2? (2)
? ?
? -
-
? -
-
-
-
-
-
? -
-
-
-
Cd2? (0.05)
?
?
-
?
-
-
?
?
?
?
?
-
-
Cd2? (0.1)
?
-
-
-
-
-
-
-
-
?
-
-
-
Cd2? (0.8)
?
-
-
-
-
-
-
-
-
-
-
-
-
T
Data were obtained in this study using the same growth conditions and methodology. Species 1, M. soli NHI-8 ; 2, M. chacoense KACC 13891T; 3, M. mediterraneum KACC 10664T; 4, M. robiniae HAMBI 3082T; 5, M. muleiense HAMBI 3264T; 6, M. temperatum HAMBI 2583T; 7, M. tamadayense LMG 26736T; 8, M. gobiense HAMBI 2974T; 9, M. tianshanense KACC 10665T; 10, M. opportunistum HAMBI 3007T; 11, M. loti KACC 11199T; 12, M. huakuii KACC 10731T; and 13, M. metallidurans LMG 24485T. w weakly positive a
The same result in the API 50CH and ID 32GN tests
b
The same result in the API 20NE test
c
The same result in the ID 32GN test
Phylogenetic analyses Genomic DNA of strain NHI-8T was isolated, and the forward primer 27F (50 -AGAGTTTGATCMTGGCTCAG-30 ) and reverse primer 1492R (50 -TACGGYTACCTTGTTACGACTT-30 ) (Frank et al. 2008) were used to amplify the 16S rRNA gene. Sequencing was performed on an Applied Biosystems 3730XL DNA analyser, using the Big Dye terminator cycle sequencing kit v.3.1 (Applied Biosystems, USA). The almostcomplete 16S rRNA gene sequence (1416 bp) of strain NHI-8T was identified by using the EzTaxon-e server (http://www.ezbiocloud.net/eztaxon) (Kim et al. 2012). The 16S rRNA gene sequences of related taxa were obtained from GenBank and edited using the BioEdit program (Hall 1999). Multiple alignments were performed with the CLUSTAL_X program (Thompson et al. 1997). Evolutionary distances were calculated using the Kimura two-parameter model (Kimura 1983). Phylogenetic trees were constructed using the neighbour-joining, maximum likelihood, and maximum parsimony methods with the MEGA v.5.03 program (Tamura et al. 2011); bootstrap values were based on 1000 replicates (Felsenstein 1985).
Mesorhizobium species; salmon sperm DNA (Invitrogen, Carlsbad, CA, USA) was used as a control. The plate was measured with a Victor X2 2030 multi-label reader (PerkinElmer, USA). The DNA G?C content was determined by HPLC, as described by Mesbah et al. (1989). Chemotaxonomic characterisation The Sherlock Microbial Identification System was used to collect cellular fatty acid data and to evaluate the relationships between species, as described by Sasser (1990). All the strains were cultured aerobically on yeast mannitol agar (YMA) at 28 °C for 3 days. Fatty acid methyl esters of strain NHI-8T and the reference type strains of recognized Mesorhizobium species were then analysed by gas chromatography (HP 6890 series GC system; Hewlett Packard, USA) using the Microbial Identification software package (v.6.08, database TSBA6). Polar lipids were extracted according to the procedures described by Minnikin et al. (1984), and alpha-naphthol was also used to detect glycolipids (Jacin and Mishkin 1965). Sequencing and phylogeny of housekeeping genes
DNA–DNA hybridization and G?C mol content DNA–DNA hybridization was carried out according to the protocol of Ezaki et al. (1989), using photobiotin-labelling DNA probes (Sigma-Aldrich) between our strain and the type strains of all recognized
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The housekeeping genes recA, glnII, and atpD can also be used to support clade grouping in rhizobial taxonomies generated with 16S rRNA sequences (Vinuesa et al. 2005; Gaunt et al. 2001; Weir et al. 2004). To amplify the atpD gene, primers 255F and
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782R were used (Vinuesa et al. 1998). The primer pairs used to amplify glnII and recA were 12F/689R and 41F/640R, respectively (Vinuesa et al. 2005). The reaction components and amplification conditions for the polymerase chain reaction (PCR) were as described previously by Vinuesa et al. (2005). Analysis of proteins by SDS-PAGE Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) can be used to distinguish between a new species and type strains (Noel and Brill 1980). It is also a useful tool for biologic analysis (Patel et al. 1988). All strains (2 9 109 cells/mL) were cultured in 10 mL of tryptone–yeast extract medium [(g L-1) tryptone, 5; yeast extract, 3; and CaCl22H2O, 0.7; pH 6.8–7.0)] at 28 °C for 2 days. Then, each culture was centrifuged at 13,000 rpm for 10 min, and the supernatant was discarded. The pellet was washed once in 10 mM Tris–HCl (pH 7.6) and then centrifuged, and the supernatant was discarded. The cells were resuspended in 0.5 mL of 10 mM Tris–HCl, pH 7.6, and then broken by ultrasonication on ice for 3 min. The concentration of protein was adjusted to achieve an optical density of 1.0 at 280 nm in an ultraviolet spectrophotometer. The samples were stored at -20 °C for up to 1 week before electrophoresis. The samples were mixed with 29 treatment buffer (5 % SDS, w/v; 30 % glycerol, v/v; 10 % 2-mercaptoethanol, v/v; 0.04 % bromophenol blue, w/v; and 20 % 1 M Tris–HCl, v/v; pH 6.8). The mixed samples were denatured at 99 °C for 5 min, and a 10 lL aliquot was loaded and electrophoresed on an SDS-polyacrylamide gel (200 9 200 mm, 1 mm thick), where a shark’s tooth comb was used for the electrophoresis. Coomassie brilliant blue R-250 (Sigma-Aldrich) was used to stain the proteins. The SDSTris–glycine buffer system was used, as described by Laemmli (1970).
Results and discussion The isolated strain was tested for growth on tryptic soy agar (TSA), R2A, YMA, nutrient agar, Luria–Bertani agar, and MacConkey agar. It grew well on TSA, unlike its related species which showed weak growth on TSA. The strain grew at 15–40 °C, and tolerated
Zn2? and Cd2? concentrations of up to 2 and 0.8 mM, respectively. A phylogenetic analysis of strain NHI-8T was performed to determine its taxonomic position in the genus Mesorhizobium. It showed a high degree of similarity with the 16S rRNA of 12 strains in the genus Mesorhizobium (97.59 % with M. chacoense PR5T; 97.38 % with M. mediterraneum UPM-Ca36T; 97.34 % with M. robiniae CCNWYC115T; 97.31 % with M. muleiense CCBAU 83963T; 97.28 % with M. temperatum SDW018T; 97.24 % with M. tamadayense Ala-3T; 97.12 % with M. gobiense CCBAU 83330T; 97.1 % with M. opportunistum WSM2075T, M. loti USDA 3471T, and M. tianshanense A-1BST; 97.08 % with M. huakuii IAM 14158T; and 97.03 % with M. metallidurans STM2683T). However, based on the neighbour-joining and maximum likelihood analyses, although the 16S rRNA sequence of NHI-8T belongs to the genus Mesorhizobium, the phyletic line is distinct and loosely associated with the group (Fig. 1). The DNA–DNA hybridization similarity values were in the range of 32.28–53.65 % only (42.49 % with M. chacoense KACC 13981T, 41.42 % with M. mediterraneum KACC 10664T, 40.05 % with M. robiniae HAMBI 3082T, 43.84 % with M. muleiense HAMBI 3264T, 42.70 % with M. temperatum HAMBI 2583T, 41.90 % with M. tamadayense LMG 26736T, 32.28 % with M. gobiense HAMBI 2974T, 48.28 % with M. tianshanense KACC 10665T, 37.68 % with M. opportunistum HAMBI 3007T, 34.51 % with M. loti KACC 11199T, 53.65 % with M. huakuii KACC 10731T, and 40.71 % with M. metallidurans LMG 24485T), which are below the value of 70 % DNA– DNA relatedness (Wayne et al. 1987), suggesting our strain is a novel member of the genus Mesorhizobium. The DNA G?C content of strain NHI-8T was determined to be 64.38 mol %. Housekeeping gene products were separated by electrophoresis on a 1.2 % agarose gel in 1 9 TE buffer with a 100 bp DNA ladder (Supplementary Fig. S1). The partial atpD, glnII, and recA fragment sequences of strain NHI-8T (approximately 405, 513, and 522 bp, respectively) were compared with those of Mesorhizobium species in GenBank, using BLAST. The atpD, glnII, and recA sequences showed similarity to the Mesorhizobium reference strains, and the closest matches were to M. chacoense STM2154T (91–97 %);
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M. mediterraneum USDA 3392T, M. muleiense CCBAU 83963T, and M. temperatum CCBAU 01545T (87–93 %); and M. chacoense LMG 19008T and M. muleiense CCBAU 83963T (90–94 %) (Fig. 2). The fatty acid profile of strain NHI-8T contained summed feature 8, cyclo-C19:0x8c, iso-C17:0, C18:0, and C16:0 components, all of which were detected in all species in this study (Supplementary Table S2) and are also specific characteristics of the genus Mesorhizobium (Tighe et al. 2000; Jarvis et al. 1996). However, there was a relatively large amount ([4 %) of C18:1x7c 11-methyl in all the strains except for strain NHI-8T (0.4 %) and M. tamadayense (not detected), which was the same in the genera Agrobacterium, Rhizobium, and Bradyrhizobium (Tighe et al. 2000). According to the description of Ramı´rez-Bahena et al. (2012), despite that M. tamadayense belongs to the genus Mesorhizobium, it showed some differences in cellular fatty acids to other Mesorhizobium species, similar to the microorganisims mentioned above. The major polar lipids detected in strain NHI-8T were cardiolipin, phosphatidylglycerol, ornithine-containing lipid, phosphatidylethanolamine, phosphatidyl-NFig. 1 Neighbour-joining phylogenetic tree, based on 16S rRNA gene sequences, showing the relationships between strain NHI-8T and related taxa. Percentage bootstrap values based on 1000 replications are given at the branch points. Bar, 0.005 substitutions per nucleotide position, where only values [50 % are given. Rhizobium leguminosarum ATCC 14480T/AY509900 was used as the outgroup. Asterisks indicate branches of the tree that were also recovered with a maximum likelihood tree
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Fig. 2 Phylogenetic tree of three concatenated gene sequences c (recA, atpD, and glnII), showing the relationships between strain NHI-8T and recognized reference strains of the genus Mesorhizobium. Percentage bootstrap values, based on 500 replications, are given at the branch points. Bar, 0.1 % nucleotide substitutions, where only values [50 % are given
dimethylethanolamine, and phosphatidylcholine (Supplementary Fig. S2), which have been detected before in the genus Mesorhizobium (Choma and Komaniecka 2002). In addition, Goldfine (1984) described all the fatty acid components in Gram-negative bacteria except for the ornithine-containing lipid, which is one of the phosphorus-free lipids and is present in eubacteria (but not in archaea and eukaryotes) (Gao et al. 2004a, b). No glycolipids were detected in strain NHI-8T when sprayed with a-naphthol-sulphuric acid at 110 °C for 15 min. The SDS-PAGE profiles of the new isolate and the reference type strains (Supplementary Fig. S3) were compared by using BioNumerics v.6.5 software (Applied Maths) with the GelComparÒ II quick guide (v.6.5; http://www.applied-maths.com). The protein profile of the new isolate showed 73–90 % similarity
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0
5
10
15
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M. chacoense KACC
13891T
M. soli NHI-8T M. gobiense HAMBI 2974T M. mediterraneum KACC 10664T M. loti KACC 11199T M. muleiense HAMBI 3264T M. temperatum HAMBI 2583T M. opportunistum HAMBI 3007T M. metallidurans LMG 24485T M. robiniae HAMBI 3082T M. huakuii KACC 10731T M. amorphae KACC 11070T M. tianshanense KACC 10665T M. tamadayense LMG 26736T
Fig. 3 Relationships among the electrophoretic protein patterns of strain NHI-8T and reference strains of Mesorhizobium species
to the reference strains, and the closest match was 90 % with M. chacoense KACC 13891T; 83 % with M. gobiense HAMBI 2974T; 81 % with M. mediterraneum KACC 10664T; 80 % with M. loti KACC 11199T; 78 % with M. muleiense HAMBI 3264T, M. temperatum HAMBI 2583T, and M. opportunistum HAMBI 3007T; 77 % with M. metallidurans LMG 24485T, M. robiniae HAMBI 3082T, and M. huakuii KACC 10731T; 75 % with M. amorphae KACC 11070T; and 73 % with M. tianshanense KACC 10665T and M. tamadayense LMG26736T. These profiles are further evidence that strain NHI-8T is a new species of the genus Mesorhizobium (Fig. 3). Based on this result and the differences in the 16S rRNA sequences described above, strain NHI-8T is considered to represent a novel species of the genus Mesorhizobium with the proposed name Mesorhizobium soli.
Description of Mesorhizobium soli sp. nov Mesorhizobium soli (so0 li. L. gen. n. soli of the soil, the source of the type strain) Gram-negative, aerobic, motile, non-spore-forming, rod-shaped bacterium. Colonies on TSA medium are
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yellow, convex, and smooth, with a diameter of 1–2 mm after 5 days at 28 °C. Cells are 0.2–0.3 lm wide and 1.3–2 lm long. The bacterium is positive for catalase and oxidase, and the hydrolysis of starch, casein, and gelatin. It is negative for methyl red and Voges–Proskauer tests and does not produce H2S. Growth occurs at pH 6–9 (optimum, pH 6.5–7.5) and in the presence of 0–2 % (w/v) NaCl (optimum, 0–0.5 %). The temperature range for growth is between 15 °C and 40 °C (optimum, 28–30 °C) on TSA. The bacterium grows well on TSA, R2A, nutrient agar, and Luria–Bertani agar, but growth is weak on MacConkey agar. In the API 50CH tests, acid production from D-/L-arabinose, D-xylose, D-mannitol, esculin ferric citrate, xylitol, D-maltose, D-saccharose, D-/L-fucose, and D-glucose is positive; and that from Dgalactose, D-fructose, D-mannose, and D-trehalose is weakly positive. No growth occurs on D-ribose, Lxylose, L-rhamnose, D-sorbitol, glycerol, N-acetylglucosamine, erythritol, D-raffinose, D-turanose, potassium gluconate, amygdalin, arbutin, salicin, D-adonitol, methyl-D-xylopyranoside, L-sorbose, L-rhamnose, dulcitol, amidon, inositol, D-cellobiose, D-tagatose, methyl-D-mannopyranoside, methyl-D-glucopyranoside, D-melibiose, D-lactose, inulin, glycogen, Dmelitose, D-/L-arabitol, gentiobiose, potassium-5-ketogluconate, and potassium-2-ketogluconate. In the API 20NE tests, assimilation of urea, esculin, Dglucose, D-mannitol, L-arabinose, D-maltose, and trisodium citrate as a sole carbon source are positive, but D-mannose is weakly assimilated; assimilation of L-arginine, N-acetylglucosamine, nitrate reduction, Ltryptophane, gelatin, 4-nitrophenyl-b-D-galactopyranoside, potassium gluconate, capric acid, adipic acid, malic acid, and phenylacetic acid are negative. In the 32 GN tests, D-saccharose, D-maltose, sodium acetate, lactic acid, D-mannitol, L-alanine, D-glucose, L-fucose, L-arabinose, trisodium citrate, and L-proline are all assimilated, whereas D-rhamnose, D-ribose, inositol, sucrose, N-acetylglucosamine, itaconic acid, suberic acid, sodium malonate, potassium-5 ketogluconate, potassium-2-ketogluconate, glycogen, 3-hydroxybutyric acid, L-serine, salicin, D-melibiose, D-sorbitol, propionic acid, capric acid, valeric acid, L-histidine, and 3- and 4-hydroxybenzoic acids are not assimilated. In the API ZYM test, the activity of leucine arylamidase, trypsin, a-glucosidase, and acid phosphatase is positive; that of esterase is weakly positive; and that of alkaline phosphatase, esterase lipase (C8),
Antonie van Leeuwenhoek
valine arylamidase, naphthol-AS-BI-phosphohydrolase, b-glucosidase, lipase (C14), cystine arylamidase, a-chymotrypsin, acid phosphatase, a-galactosidase, bgalactosidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase, and a-fucosidase is negative. The strain is sensitive to ampicillin, neomycin, and rifampicin but tolerant of the heavy metals. The strain contains summed feature 8, cycloC19:0x8c, and C18:0 as major fatty acids. The major polar lipids are cardiolipin, phosphatidylglycerol, ornithine-containing lipid, phosphatidylethanolamine, phosphatidyl-N-dimethylethanolamine, and phosphatidylcholine. The G?C content of the type strain is 64.38 mol%, as determined by HPLC. The type strain, NHI-8T (=KEMB 9005-153T = KACC 17916T = JCM 19897T), was isolated from the rhizosphere of Robinia pseudoacacia L. at Kyonggi University in Suwon, South Korea. Acknowledgments This study was supported by the Bioindustry Technology Development Program (312027-3), Minstry of Agriculture, Food and Rural Affairs, and by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science, and Technology (2011-0010144).
References Bethlenfalvay GJ, Abu-Shakra SS, Phillips DA (1978) Interdependence of nitrogen nutrition and photosynthesis in Pisum sativum L. II. Host plant response to nitrogen fixation by Rhizobium strains. Plant Physiol 62:131–133 Chen WX, Li GS, Qi YL, Wang ET, Yuan HL, Li JL (1991) Rhizobium huakuii sp. nov. isolated from the root nodules of Astragalus sinicus. Int J Syst Bacteriol 41:275–280 Chen W, Wang E, Wang S, Li Y, Chen X, Li Y (1995) Characteristics of Rhizobium tianshanense sp. nov., a moderately and slowly growing root nodule bacterium isolated from an arid saline environment in Xinjiang, People’s Republic of China. Int J Syst Bacteriol 45:153–159 Choma A, Komaniecka I (2002) Analysis of phospholipids and ornithine-containing lipids from Mesorhizobium spp. Syst Appl Microbiol 25:326–331 Doetsch RN (1981) Determinative methods of light microscopy. In: Gerdhardt P, Murray RGE, Costilow RN, Nester EW, Wood WA, Krieg NR, Phillips GB (eds) Manual of methods for general bacteriology. American Society for Microbiology, Washington, DC, pp 21–33 Ezaki T, Hashimoto Y, Yabuuchi E (1989) Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to
determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39:224–229 Felsenstein J (1985) Confidence limit on phylogenies: an approach using the bootstrap. Evolution 39:783–791 Frank JA, Reich CI, Sharma S, Weisbaum JS, Wilson BA, Olsen GJ (2008) Critical evaluation of two primers commonly used for amplification of bacterial 16S rRNA gene. Appl Environ Microbiol 74:2461–2470 Gao JL, Turner SL, Kan FL, Wang ET, Tan ZY, Qiu YH, Gu J, Terefework Z, Young JP, Lindstro¨m K, Chen WX (2004a) Mesorhizobium septentrionale sp. nov. and Mesorhizobium temperatum sp. nov., isolated from Astragalus adsurgens growing in the northern regions of China. Int J Syst Evol Microbiol 54:2003–2012 Gao JL, Weissenmayer B, Taylor AM, Thomas-Oates J, Lo´pezLara IM, Geiger O (2004b) Identification of a gene required for the formation of lyso-ornithine lipid, an intermediate in the biosynthesis of ornithine-containing lipids. Mol Microbiol 53:1757–1770 Gaunt MW, Turner SL, Rigottier-Gois L, Lloyd-Macgilp SA, Young JP (2001) Phylogenies of atpD and recA support the small subunit rRNA-based classification of rhizobia. Int J Syst Evol Microbiol 51:2037–2048 Goldfine H (1984) Bacterial membranes and lipid packing theory. J Lipid Res 25:1501–1507 Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/ NT. Nucl Acids Symp Ser 41:95–98 Han TX, Han LL, Wu LJ, Chen WF, Sui XH, Gu JG, Wang ET, Chen WX (2008) Mesorhizobium gobiense sp. nov. and Mesorhizobium tarimense sp. nov., isolated from wild legumes growing in desert soils of Xinjiang, China. Int J Syst Evol Microbiol 58:2610–2618 Howieson JG, O’Hara GW, Carr SJ (2000) Changing roles for legumes in Mediterranean agriculture: developments from an Australian perspective. Field Crops Res 65:107–122 Jacin H, Mishkin AR (1965) Separation of carbohydrates on borate impregnated silica gel G plates. J Chromatogr 18:170–173 Jarvis BDW, Pankhurst CE, Patel JJ (1982) Rhizobium loti, a new species of legume root nodule bacteria. Int J Syst Bacteriol 32:378–380 Jarvis BDW, Sivakumaran S, Tighe SW, Gillis M (1996) Identification of Agrobacterium and Rhizobium species based on cellular fatty acid composition. Plant Soil 184:143–158 Jarvis BDW, Van Berkum P, Chen WX, Nour SM, Fernandez MP, Cleyet-Marel JC, Gillis M (1997) Transfer of Rhizobium loti, Rhizobium huakuii, Rhizobium ciceri, Rhizobium mediterraneum, and Rhizobium tianshanense to Mesorhizobium gen. nov. Int J Syst Bacteriol 47:895–898 Kim OS, Cho YJ, Lee K, Yoon SH, Kim M, Na H, Park SC, Jeon YS, Lee JH, Yi H, Won S, Chun J (2012) Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol 62:716–721 Kimura M (1983) The neutral theory of molecular evolution. Cambridge University Press, Cambridge Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
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Terpolilli JJ, Hood GA, Poole PS (2012) What determines the efficiency of N2-fixing Rhizobium-legume symbioses? In: Poole RK (ed) Advances in microbial physiology, vol 60. Academic Press, London, pp 325–389 Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucl Acids Res 25:4876–4882 Tighe SW, Lajudie PD, Dipietro K, Lindstrom K, Nick G, Jarvis BDW (2000) Analysis of cellular fatty acids and phenotypic relationships of Agrobacterium, Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium species using the Sherlock microbial identification systems. Int J Syst Evol Microbiol 50:787–801 Vela´zquez E, Igual JM, Willems A, Ferna´ndez MP, Mun˜oz E, Mateos PF, Abril A, Toro N, Normand P, Cervantes E, Gillis M, Martı´nez-Molina E (2001) Mesorhizobium chacoense sp. nov., a novel species that nodulates Prosopis alba in the Chaco Arido region (Argentina). Int J Syst Evol Microbiol 51:1011–1021 Vidal C, Chantreuil C, Berge O, Maure´ L, Escarre´ J, Be´na G, Brunel B, Cleyet-Marel JC (2009) Mesorhizobium metallidurans sp. nov., a metal-resistant symbiont of Anthyllis vulneraria growing on metallicolous soil in Languedoc, France. Int J Syst Evol Microbiol 59:850–855 Vinuesa P, Rademaker JLW, Bruijn FJ, Werner D (1998) Genotypic characterization of Bradyrhizobium strains nodulating endemic woody legumes of the Canary Islands by PCR-restriction fragment length polymorphism analysis of genes encoding 16S rRNA (16S rDNA) and 16S-23S rDNA intergenic spacers, repetitive extragenic palindromic PCR genomic finger printing and partial 16S rDNA sequencing. Appl Environ Microbiol 64:2096–2104 Vinuesa P, Silva C, Werner D, Romero EM (2005) Population genetics and phylogenetic inference in bacterial molecular systematics: the roles of migration and recombination in Bradyrhizobium species cohesion and delineation. Mol Phylogenet Evol 34:29–54 Wayne LG, Brenner DJ, Colwell RR, Grimont PAD, Kandler O, Krichevsky MI, Moore LH, Moore WEC, Murray RGE, Stackebrandt E, Starr MP, Truper HG (1987) Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37:463–464 Weir BS, Turner SJ, Silvester WB, Park DC, Young JM (2004) Unexpectedly diverse Mesorhizobium strains and Rhizobium leguminosarum nodulate native legume genera of New Zealand, while introduced legume weeds are nodulated by Bradyrhizobium species. Appl Environ Microbiol 70:5980–5987 Zhang JJ, Liu TY, Chen WF, Wang ET, Sui XH, Zhang XX, Li Y, Li Y, Chen WX (2012) Mesorhizobium muleiense sp. nov., nodulating with Cicer arietinum L. Int J Syst Evol Microbiol 62:2737–2742 Zhou PF, Chen WM, Wei GH (2010) Mesorhizobium robiniae sp. nov., isolated from root nodules of Robinia pseudoacacia. Int J Syst Evol Microbiol 60:2552–2556
ORIGINAL PAPER
Mesorhizobium soli sp. nov., a novel species isolated from the rhizosphere of Robinia pseudoacacia L. in South Korea by using a modified culture method Tuan Manh Nguyen . Van Hong Thi Pham . Jaisoo Kim
Received: 19 March 2015 / Accepted: 13 May 2015 Ó Springer International Publishing Switzerland 2015
Abstract Strain NHI-8T was isolated from a forest soil sample, collected in South Korea, by using a modified culture method. Comparative analysis of its nearly full-length 16S rRNA gene sequence showed that strain NHI-8T belongs to the genus Mesorhizobium and to be closely related to Mesorhizobium chacoense PR5T (97.32 %). The levels of DNA– DNA relatedness between strain NHI-8T and reference type strains of the genus Mesorhizobium were 32.28–53.65 %. SDS-PAGE of total soluble proteins and the sequences of the housekeeping genes recA, glnII, and atpD were also used to support the clade grouping in rhizobia. The new strain contained summed feature 8 (57.0 %), cyclo-C19:0x8c (17.3 %), and C18:0 (11.0 %) as the major fatty acids, as in genus Mesorhizobium. The strain contained cardiolipin, phosphatidylglycerol, ornithinecontaining lipid, phosphatidylethanolamine,
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA, recA, atpD, and glnII genes are KC484966, KM188061, KM188059, and KM188060, respectively.
Electronic supplementary material The online version of this article (doi:10.1007/s10482-015-0481-8) contains supplementary material, which is available to authorized users. T. M. Nguyen V. H. T. Pham J. Kim (&) Department of Life Science, College of Natural Sciences, Kyonggi University, Suwon, Gyeonggi-Do 443-760, Republic of Korea e-mail: [email protected]
phosphatidyl-N-dimethylethanolamine, and phosphatidylcholine. Morphological and physiological analyses were performed to compare the characteristics of our strain with those of the reference type strains. Based on the results, strain NHI-8T was determined to represent a novel member of the genus Mesorhizobium, and the name Mesorhizobium soli is proposed. The type strain is NHI-8T (=KEMB 9005-153T = KACC 17916T = JCM 19897T). Keywords Mesorhizobium sp. nov. Housekeeping gene Taxonomy Modified culture method Rhizosphere Robinia pseudoacacia L
Introduction Bacteria are a large domain of prokaryotic microorganisms that are involved in various metabolic cycles, thus creating balance in nature. Within the nitrogen cycle, bacteria, in symbiotic association with plants, fix nitrogen, which helps to enhance host photosynthesis (Bethlenfalvay et al. 1978). One group of nitrogen-fixing bacteria, the rhizobia, are Gramnegative soil bacteria that are found in nodules on the roots of plants and fix nitrogen from the atmosphere into ammonia (Weir et al. 2004; Terpolilli et al. 2012). The host range for rhizobial species is limited, with approximately 200 known plant species that are mainly legumes (Weir et al. 2004). With the help of
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Antonie van Leeuwenhoek
rhizobial inoculants, the yields of various plants have been increased, including soybeans in the United States (Lohrke et al. 1996), European Lotus corniculatus in New Zealand (Sullivan et al. 1995), and Asian Cicer arietinum (chickpea) in Australia (Howieson et al. 2000). The genus Mesorhizobium contains many distinctive characteristics from the genus Rhizobium. Jarvis et al. (1997) re-classified and re-described the genus Mesorhizobium with major characterisations; namely, the species are Gram-negative, aerobic, nonspore-forming, rod-shaped, and motile. They often form nitrogen-fixing nodules on the roots of a limited range of legume plants (Laranjo et al. 2014). Currently, there are 30 known species (http://www.bacterio. net/mesorhizobium.html), most of which were found in China. Strain NHI-8T was isolated from the rhizosphere of Robinia pseudoacacia in South Korea by using a modified culture method. In total, 12 species were found to be closely related to strain NHI-8T, the majority of which were isolated from the legume family (Table S1). To determine the taxonomic classification of our strain, we performed DNA–DNA hybridization with related species, determined its fatty acid profile and the sequences of its housekeeping genes (recA, atpD, and glnII), and collected other information on its morphology and physiology.
Materials and methods Isolation of strain Samples were collected from the surface soil covering plant roots in the forest at Kyonggi University in Suwon, Gyeonggi-do, South Korea. Bacteria were isolated by using a modified culture method, as described by Pham and Kim (2014). Briefly, 3 g of soil was added to each well of a transwell plate, and then 3 mL of R2A medium [(g L-1); proteose peptone, 0.5; yeast extract,0.5; acid digest of casein, 0.5; glucose, 0.5; soluble starch, 0.5; dipotassium phosphate, 0.3; magnesium sulphate, 0.024; and sodium pyruvate, 0.3; pH 7.2] was added into each transwell insert. Then, 100 lL of a soil suspension (containing 1 g of soil in 10 mL of R2A medium, thoroughly stirred and allowed to settle) was inoculated in each insert. The plate was incubated in a shaking incubator at 120 rpm and 28 °C for 2 weeks. After the
123
2 weeks of incubation, serial dilutions of the culture were prepared. An aliquot (100 lL) of each dilution was spread on an R2A agar plate, and the plate was incubated at 28 °C for 5 days. Different types of colonies were picked and streaked on individual agar plates, and this was repeated until a pure colony was obtained. The pure colony was preserved in a 20 % glycerol stock at -20 °C. Phenotypic characterisation Various morphological and physiological tests were performed to compare the characteristics of our strain with those of type strains. The cell size was measured by microscopy (BX50 microscope; Olympus, Japan). Motility was assessed in tryptic soy broth medium (CM0129; Oxoid) containing 0.4 % agar. Gram staining was carried out according to the method of Doetsch (1981). Catalase activity was assessed by bubble production in 3 % (v/v) H2O2, and oxidase activity was determined by using 1 % (w/v) tetramethyl-p-phenylenediamine. Hydrolysis of starch, casein, and gelatin, hydrogen sulphide (H2S) production, and methyl red and Voges–Proskauer tests were determined as described by Smibert and Krieg (1994). Growth at different temperatures (4, 10, 15, 20, 25, 28, 30, 37, 40, 50 and 55 °C) and at various pH values (pH 4.0–12.0; at intervals of 0.5 pH unit) was determined with different buffers at 100 mM concentration (acetate buffer for pH 4–5; phosphate buffer for pH 5–8; Tris buffer for pH 9–12), and tolerance to various concentrations of NaCl (0–10 %, at 0.5 % intervals) was assessed after incubation for up to 5 days. Yeast mannitol broth medium [YMB (g L-1): manitol, 10; KH2PO4, 0.5; sodium glutamate, 0.5; NaCl, 50 9 10-3; solution A, 10 mL; solution B, 1 mL; solution C, 1 mL; yeast extract, 1; pH 6.8 (solution A: 1 g of MgSO47H2O in 100 mL of distilled water; solution B, 5.28 g of CaCl2H2O in 100 mL of distilled water; solution C, 666 9 10-3 g of FeCl36H2O in 100 mL of distilled water)] was used for determining the growth of each strain under different temperature and pH values, NaCl concentrations, and metal ion types. API 50CH (with API 50 CHB/E medium), ID 32GN, API 20NE, and API ZYM strips (bioMe´rieux, France) were used to compared phenotypic characteristics between the strain and type species of the genus Mesorhizobium (Table 1).
Antonie van Leeuwenhoek Table 1 Differential phenotypic characteristics between strain NHI-8T and closely related species of the genus Mesorhizobium Characteristic
1
2
3
4
5
6
7
8
9
10
11
12
13
?
-
-
?
?
-
-
-
?
-
-
w
?
? ?
? w
? -
? ?
? -
-
? -
w
?
? ?
? -
? -
-
Urea
?
-
-
-
-
-
?
-
-
-
?
?
-
Trisodium citratec
?
-
-
-
-
-
-
-
-
-
-
-
-
Esculin ferric citrateb
?
-
-
-
-
-
-
-
-
-
?
-
-
D-Fucose
?
?
-
-
-
-
-
-
?
-
?
-
-
?
-
?
-
-
?
?
-
w
-
?
-
-
?
?
-
?
?
-
?
?
?
-
?
?
-
D-Arabinose
?
-
?
-
?
-
?
?
?
?
w
?
-
D-Ribose
-
?
-
?
?
?
?
-
-
?
?
-
?
L-Xylose
-
-
-
-
-
-
w
-
-
-
?
-
?
D-Galactose
w
?
?
?
?
-
-
?
-
?
-
-
?
D-Fructose
w
-
?
?
?
-
-
?
w
-
-
?
?
D-Mannose
w
-
-
?
-
-
-
?
?
-
-
-
?
Assimilation of API 20NE D-Glucose
a
L-Arabinose D-Maltose
a
a
Acid from API 50CH
L-Fucose
c
D-Saccharose
c
L-Rhamnose
-
-
?
-
?
-
?
-
?
-
?
?
?
D-Sorbitol
w
-
-
? -
? -
-
-
-
? -
?
? ?
-
?
D-Trehalose
Xylitol
?
-
-
-
-
-
-
-
?
-
?
-
D-Xylose
?
-
-
?
?
w
-
?
?
-
-
?
?
D-Tagatose
-
-
-
-
-
-
-
-
?
?
-
-
-
Inositol
-
-
-
-
-
?
w
?
-
?
-
?
?
Potassium gluconate
-
-
-
w
-
-
?
-
?
-
-
-
?
Assimilation of ID 32GN Sodium acetate
?
-
-
-
?
-
?
-
-
-
-
-
Lactic acid
?
-
-
-
-
-
?
-
-
-
-
-
–
L-Alanine
?
-
-
-
-
-
-
-
-
-
-
-
-
L-Proline
?
-
-
?
-
?
?
?
w
-
-
-
?
N-Acetyl-glucosamine
-
-
-
-
-
-
?
-
?
?
-
-
-
Enzymatic activities (API ZYM) Alkaline phosphatase
-
?
?
?
?
?
w
?
w
?
?
?
?
Esterase (C4) Trypsin
w ?
w ?
? ?
? ?
? ?
? ?
? -
? ?
? ?
w ?
w ?
? w
w ?
a-Glucosidase
?
w
-
-
-
-
-
-
-
-
-
-
-
Ampicillin (0.5)
-
-
?
?
-
?
-
?
?
?
?
-
?
Neomycin (5)
-
-
?
?
-
?
-
?
-
-
-
?
?
Rifampicin (0.25)
-
-
?
?
-
?
-
?
?
?
?
?
-
Zn2? (0.1)
?
?
?
?
-
?
?
?
?
?
?
?
?
Zn2? (0.3)
?
?
?
?
-
-
?
?
?
?
?
-
?
Zn2? (0.5)
?
?
?
?
-
-
?
-
-
?
-
-
?
Resistant to (lg mL-1)
Tolerance of (mM)
123
Antonie van Leeuwenhoek Table 1 continued Characteristic
1
2
3
4
5
6
7
8
9
10
11
12
13
Zn2? (1) Zn2? (2)
? ?
? -
-
? -
-
-
-
-
-
? -
-
-
-
Cd2? (0.05)
?
?
-
?
-
-
?
?
?
?
?
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Data were obtained in this study using the same growth conditions and methodology. Species 1, M. soli NHI-8 ; 2, M. chacoense KACC 13891T; 3, M. mediterraneum KACC 10664T; 4, M. robiniae HAMBI 3082T; 5, M. muleiense HAMBI 3264T; 6, M. temperatum HAMBI 2583T; 7, M. tamadayense LMG 26736T; 8, M. gobiense HAMBI 2974T; 9, M. tianshanense KACC 10665T; 10, M. opportunistum HAMBI 3007T; 11, M. loti KACC 11199T; 12, M. huakuii KACC 10731T; and 13, M. metallidurans LMG 24485T. w weakly positive a
The same result in the API 50CH and ID 32GN tests
b
The same result in the API 20NE test
c
The same result in the ID 32GN test
Phylogenetic analyses Genomic DNA of strain NHI-8T was isolated, and the forward primer 27F (50 -AGAGTTTGATCMTGGCTCAG-30 ) and reverse primer 1492R (50 -TACGGYTACCTTGTTACGACTT-30 ) (Frank et al. 2008) were used to amplify the 16S rRNA gene. Sequencing was performed on an Applied Biosystems 3730XL DNA analyser, using the Big Dye terminator cycle sequencing kit v.3.1 (Applied Biosystems, USA). The almostcomplete 16S rRNA gene sequence (1416 bp) of strain NHI-8T was identified by using the EzTaxon-e server (http://www.ezbiocloud.net/eztaxon) (Kim et al. 2012). The 16S rRNA gene sequences of related taxa were obtained from GenBank and edited using the BioEdit program (Hall 1999). Multiple alignments were performed with the CLUSTAL_X program (Thompson et al. 1997). Evolutionary distances were calculated using the Kimura two-parameter model (Kimura 1983). Phylogenetic trees were constructed using the neighbour-joining, maximum likelihood, and maximum parsimony methods with the MEGA v.5.03 program (Tamura et al. 2011); bootstrap values were based on 1000 replicates (Felsenstein 1985).
Mesorhizobium species; salmon sperm DNA (Invitrogen, Carlsbad, CA, USA) was used as a control. The plate was measured with a Victor X2 2030 multi-label reader (PerkinElmer, USA). The DNA G?C content was determined by HPLC, as described by Mesbah et al. (1989). Chemotaxonomic characterisation The Sherlock Microbial Identification System was used to collect cellular fatty acid data and to evaluate the relationships between species, as described by Sasser (1990). All the strains were cultured aerobically on yeast mannitol agar (YMA) at 28 °C for 3 days. Fatty acid methyl esters of strain NHI-8T and the reference type strains of recognized Mesorhizobium species were then analysed by gas chromatography (HP 6890 series GC system; Hewlett Packard, USA) using the Microbial Identification software package (v.6.08, database TSBA6). Polar lipids were extracted according to the procedures described by Minnikin et al. (1984), and alpha-naphthol was also used to detect glycolipids (Jacin and Mishkin 1965). Sequencing and phylogeny of housekeeping genes
DNA–DNA hybridization and G?C mol content DNA–DNA hybridization was carried out according to the protocol of Ezaki et al. (1989), using photobiotin-labelling DNA probes (Sigma-Aldrich) between our strain and the type strains of all recognized
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The housekeeping genes recA, glnII, and atpD can also be used to support clade grouping in rhizobial taxonomies generated with 16S rRNA sequences (Vinuesa et al. 2005; Gaunt et al. 2001; Weir et al. 2004). To amplify the atpD gene, primers 255F and
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782R were used (Vinuesa et al. 1998). The primer pairs used to amplify glnII and recA were 12F/689R and 41F/640R, respectively (Vinuesa et al. 2005). The reaction components and amplification conditions for the polymerase chain reaction (PCR) were as described previously by Vinuesa et al. (2005). Analysis of proteins by SDS-PAGE Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) can be used to distinguish between a new species and type strains (Noel and Brill 1980). It is also a useful tool for biologic analysis (Patel et al. 1988). All strains (2 9 109 cells/mL) were cultured in 10 mL of tryptone–yeast extract medium [(g L-1) tryptone, 5; yeast extract, 3; and CaCl22H2O, 0.7; pH 6.8–7.0)] at 28 °C for 2 days. Then, each culture was centrifuged at 13,000 rpm for 10 min, and the supernatant was discarded. The pellet was washed once in 10 mM Tris–HCl (pH 7.6) and then centrifuged, and the supernatant was discarded. The cells were resuspended in 0.5 mL of 10 mM Tris–HCl, pH 7.6, and then broken by ultrasonication on ice for 3 min. The concentration of protein was adjusted to achieve an optical density of 1.0 at 280 nm in an ultraviolet spectrophotometer. The samples were stored at -20 °C for up to 1 week before electrophoresis. The samples were mixed with 29 treatment buffer (5 % SDS, w/v; 30 % glycerol, v/v; 10 % 2-mercaptoethanol, v/v; 0.04 % bromophenol blue, w/v; and 20 % 1 M Tris–HCl, v/v; pH 6.8). The mixed samples were denatured at 99 °C for 5 min, and a 10 lL aliquot was loaded and electrophoresed on an SDS-polyacrylamide gel (200 9 200 mm, 1 mm thick), where a shark’s tooth comb was used for the electrophoresis. Coomassie brilliant blue R-250 (Sigma-Aldrich) was used to stain the proteins. The SDSTris–glycine buffer system was used, as described by Laemmli (1970).
Results and discussion The isolated strain was tested for growth on tryptic soy agar (TSA), R2A, YMA, nutrient agar, Luria–Bertani agar, and MacConkey agar. It grew well on TSA, unlike its related species which showed weak growth on TSA. The strain grew at 15–40 °C, and tolerated
Zn2? and Cd2? concentrations of up to 2 and 0.8 mM, respectively. A phylogenetic analysis of strain NHI-8T was performed to determine its taxonomic position in the genus Mesorhizobium. It showed a high degree of similarity with the 16S rRNA of 12 strains in the genus Mesorhizobium (97.59 % with M. chacoense PR5T; 97.38 % with M. mediterraneum UPM-Ca36T; 97.34 % with M. robiniae CCNWYC115T; 97.31 % with M. muleiense CCBAU 83963T; 97.28 % with M. temperatum SDW018T; 97.24 % with M. tamadayense Ala-3T; 97.12 % with M. gobiense CCBAU 83330T; 97.1 % with M. opportunistum WSM2075T, M. loti USDA 3471T, and M. tianshanense A-1BST; 97.08 % with M. huakuii IAM 14158T; and 97.03 % with M. metallidurans STM2683T). However, based on the neighbour-joining and maximum likelihood analyses, although the 16S rRNA sequence of NHI-8T belongs to the genus Mesorhizobium, the phyletic line is distinct and loosely associated with the group (Fig. 1). The DNA–DNA hybridization similarity values were in the range of 32.28–53.65 % only (42.49 % with M. chacoense KACC 13981T, 41.42 % with M. mediterraneum KACC 10664T, 40.05 % with M. robiniae HAMBI 3082T, 43.84 % with M. muleiense HAMBI 3264T, 42.70 % with M. temperatum HAMBI 2583T, 41.90 % with M. tamadayense LMG 26736T, 32.28 % with M. gobiense HAMBI 2974T, 48.28 % with M. tianshanense KACC 10665T, 37.68 % with M. opportunistum HAMBI 3007T, 34.51 % with M. loti KACC 11199T, 53.65 % with M. huakuii KACC 10731T, and 40.71 % with M. metallidurans LMG 24485T), which are below the value of 70 % DNA– DNA relatedness (Wayne et al. 1987), suggesting our strain is a novel member of the genus Mesorhizobium. The DNA G?C content of strain NHI-8T was determined to be 64.38 mol %. Housekeeping gene products were separated by electrophoresis on a 1.2 % agarose gel in 1 9 TE buffer with a 100 bp DNA ladder (Supplementary Fig. S1). The partial atpD, glnII, and recA fragment sequences of strain NHI-8T (approximately 405, 513, and 522 bp, respectively) were compared with those of Mesorhizobium species in GenBank, using BLAST. The atpD, glnII, and recA sequences showed similarity to the Mesorhizobium reference strains, and the closest matches were to M. chacoense STM2154T (91–97 %);
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M. mediterraneum USDA 3392T, M. muleiense CCBAU 83963T, and M. temperatum CCBAU 01545T (87–93 %); and M. chacoense LMG 19008T and M. muleiense CCBAU 83963T (90–94 %) (Fig. 2). The fatty acid profile of strain NHI-8T contained summed feature 8, cyclo-C19:0x8c, iso-C17:0, C18:0, and C16:0 components, all of which were detected in all species in this study (Supplementary Table S2) and are also specific characteristics of the genus Mesorhizobium (Tighe et al. 2000; Jarvis et al. 1996). However, there was a relatively large amount ([4 %) of C18:1x7c 11-methyl in all the strains except for strain NHI-8T (0.4 %) and M. tamadayense (not detected), which was the same in the genera Agrobacterium, Rhizobium, and Bradyrhizobium (Tighe et al. 2000). According to the description of Ramı´rez-Bahena et al. (2012), despite that M. tamadayense belongs to the genus Mesorhizobium, it showed some differences in cellular fatty acids to other Mesorhizobium species, similar to the microorganisims mentioned above. The major polar lipids detected in strain NHI-8T were cardiolipin, phosphatidylglycerol, ornithine-containing lipid, phosphatidylethanolamine, phosphatidyl-NFig. 1 Neighbour-joining phylogenetic tree, based on 16S rRNA gene sequences, showing the relationships between strain NHI-8T and related taxa. Percentage bootstrap values based on 1000 replications are given at the branch points. Bar, 0.005 substitutions per nucleotide position, where only values [50 % are given. Rhizobium leguminosarum ATCC 14480T/AY509900 was used as the outgroup. Asterisks indicate branches of the tree that were also recovered with a maximum likelihood tree
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Fig. 2 Phylogenetic tree of three concatenated gene sequences c (recA, atpD, and glnII), showing the relationships between strain NHI-8T and recognized reference strains of the genus Mesorhizobium. Percentage bootstrap values, based on 500 replications, are given at the branch points. Bar, 0.1 % nucleotide substitutions, where only values [50 % are given
dimethylethanolamine, and phosphatidylcholine (Supplementary Fig. S2), which have been detected before in the genus Mesorhizobium (Choma and Komaniecka 2002). In addition, Goldfine (1984) described all the fatty acid components in Gram-negative bacteria except for the ornithine-containing lipid, which is one of the phosphorus-free lipids and is present in eubacteria (but not in archaea and eukaryotes) (Gao et al. 2004a, b). No glycolipids were detected in strain NHI-8T when sprayed with a-naphthol-sulphuric acid at 110 °C for 15 min. The SDS-PAGE profiles of the new isolate and the reference type strains (Supplementary Fig. S3) were compared by using BioNumerics v.6.5 software (Applied Maths) with the GelComparÒ II quick guide (v.6.5; http://www.applied-maths.com). The protein profile of the new isolate showed 73–90 % similarity
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0
5
10
15
20
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M. chacoense KACC
13891T
M. soli NHI-8T M. gobiense HAMBI 2974T M. mediterraneum KACC 10664T M. loti KACC 11199T M. muleiense HAMBI 3264T M. temperatum HAMBI 2583T M. opportunistum HAMBI 3007T M. metallidurans LMG 24485T M. robiniae HAMBI 3082T M. huakuii KACC 10731T M. amorphae KACC 11070T M. tianshanense KACC 10665T M. tamadayense LMG 26736T
Fig. 3 Relationships among the electrophoretic protein patterns of strain NHI-8T and reference strains of Mesorhizobium species
to the reference strains, and the closest match was 90 % with M. chacoense KACC 13891T; 83 % with M. gobiense HAMBI 2974T; 81 % with M. mediterraneum KACC 10664T; 80 % with M. loti KACC 11199T; 78 % with M. muleiense HAMBI 3264T, M. temperatum HAMBI 2583T, and M. opportunistum HAMBI 3007T; 77 % with M. metallidurans LMG 24485T, M. robiniae HAMBI 3082T, and M. huakuii KACC 10731T; 75 % with M. amorphae KACC 11070T; and 73 % with M. tianshanense KACC 10665T and M. tamadayense LMG26736T. These profiles are further evidence that strain NHI-8T is a new species of the genus Mesorhizobium (Fig. 3). Based on this result and the differences in the 16S rRNA sequences described above, strain NHI-8T is considered to represent a novel species of the genus Mesorhizobium with the proposed name Mesorhizobium soli.
Description of Mesorhizobium soli sp. nov Mesorhizobium soli (so0 li. L. gen. n. soli of the soil, the source of the type strain) Gram-negative, aerobic, motile, non-spore-forming, rod-shaped bacterium. Colonies on TSA medium are
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yellow, convex, and smooth, with a diameter of 1–2 mm after 5 days at 28 °C. Cells are 0.2–0.3 lm wide and 1.3–2 lm long. The bacterium is positive for catalase and oxidase, and the hydrolysis of starch, casein, and gelatin. It is negative for methyl red and Voges–Proskauer tests and does not produce H2S. Growth occurs at pH 6–9 (optimum, pH 6.5–7.5) and in the presence of 0–2 % (w/v) NaCl (optimum, 0–0.5 %). The temperature range for growth is between 15 °C and 40 °C (optimum, 28–30 °C) on TSA. The bacterium grows well on TSA, R2A, nutrient agar, and Luria–Bertani agar, but growth is weak on MacConkey agar. In the API 50CH tests, acid production from D-/L-arabinose, D-xylose, D-mannitol, esculin ferric citrate, xylitol, D-maltose, D-saccharose, D-/L-fucose, and D-glucose is positive; and that from Dgalactose, D-fructose, D-mannose, and D-trehalose is weakly positive. No growth occurs on D-ribose, Lxylose, L-rhamnose, D-sorbitol, glycerol, N-acetylglucosamine, erythritol, D-raffinose, D-turanose, potassium gluconate, amygdalin, arbutin, salicin, D-adonitol, methyl-D-xylopyranoside, L-sorbose, L-rhamnose, dulcitol, amidon, inositol, D-cellobiose, D-tagatose, methyl-D-mannopyranoside, methyl-D-glucopyranoside, D-melibiose, D-lactose, inulin, glycogen, Dmelitose, D-/L-arabitol, gentiobiose, potassium-5-ketogluconate, and potassium-2-ketogluconate. In the API 20NE tests, assimilation of urea, esculin, Dglucose, D-mannitol, L-arabinose, D-maltose, and trisodium citrate as a sole carbon source are positive, but D-mannose is weakly assimilated; assimilation of L-arginine, N-acetylglucosamine, nitrate reduction, Ltryptophane, gelatin, 4-nitrophenyl-b-D-galactopyranoside, potassium gluconate, capric acid, adipic acid, malic acid, and phenylacetic acid are negative. In the 32 GN tests, D-saccharose, D-maltose, sodium acetate, lactic acid, D-mannitol, L-alanine, D-glucose, L-fucose, L-arabinose, trisodium citrate, and L-proline are all assimilated, whereas D-rhamnose, D-ribose, inositol, sucrose, N-acetylglucosamine, itaconic acid, suberic acid, sodium malonate, potassium-5 ketogluconate, potassium-2-ketogluconate, glycogen, 3-hydroxybutyric acid, L-serine, salicin, D-melibiose, D-sorbitol, propionic acid, capric acid, valeric acid, L-histidine, and 3- and 4-hydroxybenzoic acids are not assimilated. In the API ZYM test, the activity of leucine arylamidase, trypsin, a-glucosidase, and acid phosphatase is positive; that of esterase is weakly positive; and that of alkaline phosphatase, esterase lipase (C8),
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valine arylamidase, naphthol-AS-BI-phosphohydrolase, b-glucosidase, lipase (C14), cystine arylamidase, a-chymotrypsin, acid phosphatase, a-galactosidase, bgalactosidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase, and a-fucosidase is negative. The strain is sensitive to ampicillin, neomycin, and rifampicin but tolerant of the heavy metals. The strain contains summed feature 8, cycloC19:0x8c, and C18:0 as major fatty acids. The major polar lipids are cardiolipin, phosphatidylglycerol, ornithine-containing lipid, phosphatidylethanolamine, phosphatidyl-N-dimethylethanolamine, and phosphatidylcholine. The G?C content of the type strain is 64.38 mol%, as determined by HPLC. The type strain, NHI-8T (=KEMB 9005-153T = KACC 17916T = JCM 19897T), was isolated from the rhizosphere of Robinia pseudoacacia L. at Kyonggi University in Suwon, South Korea. Acknowledgments This study was supported by the Bioindustry Technology Development Program (312027-3), Minstry of Agriculture, Food and Rural Affairs, and by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science, and Technology (2011-0010144).
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