International Journal of Systematic and Evolutionary Microbiology (2014), 64, 1218–1222
DOI 10.1099/ijs.0.055574-0
Solimonas terrae sp. nov., isolated from soil Soo-Jin Kim,1 Ji-Young Moon,1 Hang-Yeon Weon,1 Jae-Hyung Ahn,1 Wen-Ming Chen2 and Soon-Wo Kwon1 Correspondence Soon-Wo Kwon
1
[email protected]
2
Agricultural Microbiology Division, National Academy of Agricultural Science, Rural Development Administration, Suwon 441-707, Republic of Korea Laboratory of Microbiology, Department of Seafood Science, National Kaohsiung Marine University, No. 142, Hai-Chuan Rd, Nan-Tzu, Kaohsiung City 811, Taiwan, ROC
A Gram-stain-negative, aerobic, motile, rod-shaped bacterium, designated strain KIS83-12T, was isolated from soil of Gaui island in the Taean region of South Korea. The strain grew at 15–33 6C (optimum, 28 6C), at pH 5.0–8.0 (optimum, pH 7.0). Growth did not occur in the presence of NaCl. The strain was catalase-negative and oxidase-positive. Phylogenetic analysis based on 16S rRNA gene sequences showed that KIS83-12T was most closely related to Solimonas soli DCY12T (96.9 %), Solimonas variicoloris MN28T (96.5 %), Solimonas flava CW-KD 4T (96.5 %) and Solimonas aquatica NAA16T (96.0 %), and formed a robust phyletic lineage with members of the genus Solimonas. The main isoprenoid quinone was Q-8. Major polar lipids included phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. Fatty acids present in large and moderate amounts (.5.0 %) were summed feature 8 (C18 : 1v7c and/or C18 : 1v6c), C16 : 0, C16 : 1v5c, summed feature 2 (iso-C16 : 1 I and/or C14 : 0 3-OH) and C12 : 0. The DNA G+C content was 67.9 mol%. On the basis of the taxonomic data obtained in this study, KIS83-12T represents a novel species of the genus Solimonas, for which the name Solimonas terrae sp. nov. is proposed, with KIS83-12T (5KACC 16967T5DSM 27281T) as the type strain.
The family Solimonadaceae consists of two genera, Solimonas and Fontimonas (Losey et al., 2013). The genus Solimonas was proposed by Kim et al. (2007) for Gramnegative, non-motile, rod-shaped, oxidase-negative and catalase-positive bacteria isolated from soil in a ginseng field. Later, three more species were added to the genus Solimonas. Isolates from freshwater spring were named Solimonas aquatica, Sinobacter flavus and Singularimonas variicoloris and were reclassified as Solimonas flava and Solimonas variicoloris, respectively (Sheu et al., 2011). The genus Solimonas was emended as Gram-negative, rod-shaped, aerobic or facultatively anaerobic, oxidase- and catalasepositive, and motile or non-motile bacteria. Members of the genus Solimonas are characterized by having C16 : 0 and C18 : 1v7c as their major fatty acids, and phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and an uncharacterized aminophospholipid as predominant polar lipids. The main respiratory isoprenoid quinone is Q-8. Bacterial strain KIS83-12T was isolated from the soil of Gaui island in the Taean region of South Korea, using the standard dilution plating technique. Isolation was achieved using a R2A (Difco), and the isolate was routinely cultured The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain KIS83-12T is KF112046. Two supplementary figures are available with the online version of this paper.
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on R2A (Difco) agar at 28 uC and maintained as 15 % (v/v) glycerol suspensions at 280 uC. The 16S rRNA gene of strain KIS83-12T was amplified using the universal primers 9F and 1512R (Weisburg et al., 1991), and was sequenced by Genotec, Daejeon, South Korea. Its sequence was 1433 bp and was compared with other sequences in the EzTaxon-e server (http://eztaxon-e. ezbiocloud.net; Kim et al., 2012). The 16S rRNA gene sequences of strain KIS83-12T and associated species were aligned using the integrated SINA alignment tool from the ARB-SILVA website (Pruesse et al., 2007). Phylogenetic trees based on aligned sequences and evolutionary analyses were reconstructed using MEGA version 5 (Tamura et al., 2011) with three algorithms: neighbour-joining (Saitou & Nei, 1987), maximum-parsimony (Kluge & Farris, 1969) and maximum-likelihood (Felsenstein, 1981) based on 1000 randomly chosen bootstrap replications. Strain KIS83-12T showed highest sequence similarities to the species of the genus Solimonas, Solimonas soli DCY12T (96.9 %), Solimonas variicoloris MN28T (96.5 %), Solimonas flava CW-KD 4T (96.5 %) and Solimonas aquatica NAA16T (96.0 %). Its degree of sequence similarity with other species with validly published names was less than 94.0 %. The neighbourjoining phylogenetic tree revealed that strain KIS83-12T clustered with members of the genus Solimonas; this was supported by the maximum-likelihood and maximumparsimony trees (Fig. 1). 055574 G 2014 IUMS Printed in Great Britain
Solimonas terrae sp. nov.
0.02
97 85
95
Solimonas variicoloris MN 28T (AJ555478) Solimonas flava CW-KD 4T (EF154515) Solimonas soli DCY12T (EF067861)
100
Solimonas terrae KIS83-12T (KF112046)
100
Solimonas aquatica NAA16T (EU303271) Fontimonas thermophila HA-01T (JN415769)
100
Alkanibacter difficilis MN154.3T (AJ313020) Nevskia soli GR15-1T (EF178286)
100
100
Nevskia ramosa Soe1T (AJ001010) Hydrocarboniphaga effusa AP103T (AY363245) 99
100
Hydrocarboniphaga daqingensis B2-9T (EU313812) Rhodanobacter ginsengisoli GR17-7T (EF166075) Dyella japonica XD53T (AB110498)
Arenimonas malthae CC-JY-1T (DQ239766)
99
Thermomonas haemolytica A50-7-3T (AJ300185)
93
Luteimonas mephitis B1953/27.1T (AJ012228)
98
Lysobacter soli DCY21T (EF623862)
87 97
Xanthomonas melonis LMG 8670T (Y10756) Pseudoxanthomonas yeongjuensis GR12-1T (DQ438977) Escherichia coli DSM 30083T (X80725)
Fig. 1. Neighbour-joining phylogenetic tree showing the position of strain KIS83-12T, based on an alignment of 16S rRNA gene sequences. Filled circles indicate that the corresponding branches were also recovered both in the maximum-parsimony and maximum-likelihood trees. Bootstrap percentages (based on 1000 replications) .70 % are shown at branching points. Bar, 0.02 substitutions per nucleotide position.
Strain KIS83-12T was tested for a range of phenotypic, physiological and biochemical characteristics together with type strains of species of the genus Solimonas. The morphology of cells grown on R2A agar at 28 uC for 2 days, after negative staining with 1 % (w/v) sodium phosphotungstic acid (pH 7.0) was investigated with an electron microscope (LEO model 912AB). Gram staining was determined using heat-fixed liquid cultures and a Difco Gram-staining kit, according to the manufacturer’s instructions. Physiological and biochemical properties were tested with API 20NE, API ID 32GN and API ZYM test kits (bioMe´rieux). The test procedure followed the manufacturer’s instructions except that assimilation results were checked after an incubation period of up to 14 days. Catalase activity was assessed by bubble production in 3 % (v/v) H2O2 and oxidase activity was determined using 1 % (w/v) tetramethyl-p-phenylenediamine. Growth at 4, 10, 15, 20, 25, 28, 30, 33, 35, 37 and 40 uC was assessed after 14 days on R2A agar. Growth at pH 4.0–10.0 (at intervals of 1.0 pH unit) was assessed after 14 days in R2A broth; pH was adjusted with citrate/phosphate buffer, Tris/HCl buffer, HCl or NaOH (Breznak & Costilow, 1994). Growth in 0, 0.5, 1, 2 and 3 % (w/v) NaCl was assessed after 14 days in R2A broth. Anaerobic growth was investigated using incubation in the BBL GasPak Anaerobic System (Difco) for 7 days at 28 uC on R2A agar supplemented with 0.5 % (w/v) Na2SO4, 0.5 % (w/v) NaNO3, 0.5 % (w/v) NaHCO3, http://ijs.sgmjournals.org
or 0.02 % (w/v) FeCl3. Casein, starch and tyrosine degradation was examined on R2A plates containing milk powder (5 %, w/v), starch (1 %, w/v) or tyrosine (0.1 %, w/v), respectively. The degradation of CM-cellulose and Tween 80 was examined using R2A supplemented with 1 % (w/v) substrates. DNase activity was determined with DNase test agar (Difco). For antibiotic testing, antimicrobial compound discs (BD) were used as follows: amikacin (30 mg), ampicillin (10 mg), cefotaxime (30 mg), cephalothin (30 mg), chloramphenicol (30 mg), ciprofloxacin (5 mg), erythromycin (15 mg), gentamicin (10 mg), imipenem (10 mg), kanamycin (30 mg), nalidixic acid (30 mg), netilmicin (30 mg), penicillin (10 mg), polymyxin (300 IU), rifampicin (5 mg), streptomycin (10 mg), tetracycline (30 mg) and vancomycin (30 mg). The effect of antimicrobial compounds on cell growth was assessed after 4 days at 30 uC. The diameter of the antibiotic discs was 8 mm. A strain was considered susceptible when the diameter of the inhibition zone was .13 mm, intermediate at 10–12 mm and resistant at ,10 mm. Strain KIS83-12T was aerobic, Gram-stain-negative, rod-shaped, non-spore-forming and motile by means of a single polar flagellum (Fig. S1 available in the online Supplementary Material). Growth occurred at 15–33 uC (optimum, 28 uC), at pH 5.0–8.0 (optimum, pH 7.0). Growth did not occur in the presence of NaCl. Strain KIS83-12T grew on R2A, weakly on nutrient agar (Difco), but not on trypticase soy agar (Difco) or McConkey 1219
S.-J. Kim and others
Table 1. Phenotypic comparison of strain KIS83-12T and species of the genus Solimonas Strain: 1, KIS83-12T; 2, Solimonas aquatica NAA 16T; 3, Solimonas flava CW-KD 4T; 4, Solimonas soli DCY 12T; 5, Solimonas variicoloris MN 28T. All strains are positive for aesculin hydrolysis and oxidase, but negative for nitrate reduction, indole production, glucose fermentation and arginine dihydrolase. None of the strains assimilate D-mannose, potassium gluconate, trisodium citrate or phenylacetic acid. All strains showed positive activities for esterase lipase (C8), acid phosphatase and naphthol-AS-BI-phosphohydrolase, but negative activities for lipase (C14), trypsin, achymotrypsin, a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase. +, Positive; 2, negative; W, weakly positive. Characteristic Isolation source Colony colour Cell morphology Cell size (mm) Motility Catalase Temperature range (uC) Growth on 1 % NaCl (w/v) DNA hydrolysis Urease Gelatin hydrolysis b-Galactosidase Assimilation of: D-Glucose L-Arabinose D-Mannitol N-Acetylglucosamine Maltose Capric acid Adipic acid Malic acid Enzymic activity of: Alkaline phosphatase Esterase (C4) Leucine arylamidase Valine arylamidase Cystine arylamidase b-Glucosidase Antibiotic susceptibility to: Ampicillin (10 mg) Cephalothin (30 mg) Imipenem (10 mg) Penicillin (10 mg) Polymyxin (300 IU) DNA G+C content (mol%)
1
2
3
4
Water * White Short rod 0.6–0.861.0–1.6 +a – 20–40a +a –a + – –
Soil Yellow Rod 0.5–0.661.3–1.8 –a + 15–42a +a –a – + –
Soil Light yellow Rod 0.5–0.661.3–1.9 +a + 20–42a +a +a – – –
Biofilterd Yellow Long rod 0.4–0.561.6–3.1 –a + 20–42a +a –a – + –
– – – – – – – –
+ + + + + – + –
+ – + – – + – +
– – – – – – – –
– – – – – – – –
+ + + – + –
– – + + – –
+ + + – – –
+ + – – – –
+ + – – – +
+ – + +
+ + – – – 66.2a
– – + – + 65.1b
+ – + +
+ + + + + 64.9d
W
b
5
Soil Light yellow Long rod 0.5–0.661.5–2.4 + – 15–33 – – – + +
67.9
a
c
W
68.4a
*Data from: a, Sheu et al. (2011); b, Zhou et al., (2008); c, Kim et al., (2007); d, Friedrich & Lipski, (2008).
For fatty acid analysis, strain KIS83-12T and the four reference strains were grown on R2A at 28 uC for 48 h to the
late exponential phase. The fatty acid methyl esters were obtained by saponification, methylation and extraction, and analysed using the Sherlock Microbial Identification System (MIDI) according to the manufacturer’s instructions. Fatty acid methyl esters were analysed by using the Microbial Identification software package (Sherlock Version 6.1; MIDI database: TSBA6). Polar lipids were examined using twodimensional TLC. The reagents used for detection of polar lipids were as follows: 5 % (v/v) ethanolic molybdophosphoric acid was used for detection of all lipids, ninhydrin
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International Journal of Systematic and Evolutionary Microbiology 64
Agar (Difco). Table 1 shows differential characteristics of KIS83-12T and other strains of species of the genus Solimonas. Strain KIS83-12T could be differentiated from species of the genus Solimonas with validly published names on the basis of morphology, catalase activity, temperature range for growth, NaCl tolerance, ability to hydrolyse several substrates, assimilation patterns and sensitivity to several antibiotics.
Solimonas terrae sp. nov.
reagent for lipids containing free amino groups, Zinzadze reagent for phosphorus-containing lipids and a-naphthol reagent for glycolipids (Collins et al., 1980). Quinones were characterized as described by Minnikin et al. (1984). The G+C content was determined by the fluorometric method (Gonzalez & Saiz-Jimenez, 2002) using SYBR Green 1 and a real-time PCR thermocycler (Bio-Rad). Strain KIS83-12T contained large and moderate amounts of the fatty acids summed feature 8 (C18 : 1v7c and/or C18 : 1v6c; 39.8 %), C16 : 0 (15.6 %), C16 : 1v5c (10.3 %), summed feature 2 (isoC16 : 1 I and/or C14 : 0 3-OH; 5.6 %) and C12 : 0 (5.0 %), along with smaller amounts of several fatty acids (Table 2). These fatty acids present in strain KIS83-12T in large and moderate amounts were detected in other species of the genus
Table 2. Cellular fatty acid compositions (%) of strain KIS8312T and the type strains of species of the genus Solimonas Strain: 1, KIS83-12T; 2, Solimonas aquatica NAA 16T; 3, Solimonas flava CW-KD 4T; 4, Solimonas soli DCY 12T; 5, Solimonas variicoloris MN 28T. All fatty acid data were obtained in this study from cells grown on R2A for 48 h. –, not detected or ,0.5% of the total fatty acids. Fatty acid C10 : 0 C12 : 0 iso-C12 : 0 C12 : 0 2-OH C12 : 0 3-OH C14 : 0 iso-C14 : 0 3-OH C14 : 0 2-OH C16 : 0 iso-C16 : 0 C16 : 0 2-OH C16 : 0 3-OH C16 : 1v5c C16 : 1v11c C16 : 1v7c alcohol C17 : 0 C17 : 1v6c C17 : 0 cyclo 10-Methyl C17 : 0 C18 : 0 iso-C18 : 1 H C19 : 0 cyclo v8c Summed feature* 1 2 3 8
1
2
3
4
5
2.2 5.0 – – 0.8 4.7 – 1.4 15.6 3.1 – – 10.3 2.5 0.6 – – – 1.8 – 0.7 –
– 6.9 0.7 – – 4.3 – – 18.2 5.2 – 0.9 6.5 – – – – 0.7 – – 0.7 –
– 3.7 1.2 2.2 1.2 5.5 1.1 – 13.3 7.3 – – 6.2 5.4 – – – – – – 1.2 –
– 4.4 – 3.0 – 5.2 – – 11.5 – 0.8 – 12.1 – – 1.4 0.8 – – – – 2.0
– 5.5 0.6 1.6 – 5.4 – – 15.2 2.2 – – 3.7 4.1 – – – – – 0.7 – –
– 5.6 3.6 39.8
– 6.9 23.3 23.8
– 7.9 3.3 37.3
1.3 9.4 4.0 41.3
– 9.1 3.7 45.8
*Summed features are combinations of fatty acids that cannot be separated by the MIDI system. Summed feature 1 comprises C13 : 0 3OH and/or iso-C15 : 1 H; summed feature 2 comprises iso-C16 : 1 I and/ or C14 : 0 3-OH; summed feature 3 comprises C16 : 1v7c and/or C16 : 1v6c; summed feature 8 comprises C18 : 1v7c and/or C18 : 1v6c. http://ijs.sgmjournals.org
Solimonas, despite small quantitative differences. Strain KIS83-12T contained large amounts of phosphatidylethanolamine (PE), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG) as the major polar lipids and it also contained a small amount of aminophospholipid and small amounts of several aminolipids (Fig. S2). The polar lipid profiles of other strains of species of the genus Solimonas indicated that aminophospholipids along with PE, PG and DPG were major polar lipids (Sheu et al., 2011). However, strain KIS83-12T contained only a small amount of aminophospholipid. Strain KIS83-12T contained Q-8 as the predominant respiratory quinone, which is in accordance with the genus Solimonas (Sheu et al., 2011). The genomic DNA G+C content of strain KIS83-12T was 67.9 mol%. It is evident, based on a 16S rRNA gene sequence similarity of less than 97 % (,96.9 %) with species with validly published names, a phylogenetic clustering with other species of the genus Solimonas (Fig. 1) and differential taxonomic characteristics (Table 1), that strain KIS83-12T should be classified as a novel species, distinct from currently recognized species of the genus Solimonas, and the name Solimonas terrae sp. nov. is proposed. Description of Solimonas terrae sp. nov. Solimonas terrae (ter9rae. L. gen. n. terrae of the soil). Cells are aerobic, Gram-stain-negative, motile rods, 0.5– 0.6 mm61.5–2.4 mm. Catalase-negative and oxidase-positive. Grows on R2A, weakly on nutrient agar, but not on trypticase soy agar or McConkey agar. Colonies are light yellow, convex, round with entire edges on R2A agar at 28 uC. Grows in the temperature range of 15–33 uC (optimum, 28 uC) and at pH5.0–8.0 (optimum 7.0). Grows without NaCl. Hydrolyses tyrosine, but not casein, cellulose, chitin, DNA, hypoxanthine, Tween 80, starch or xanthine. With API 20NE, positive for aesculin hydrolysis, gelatin hydrolysis and b-galactosidase (PNG), but negative for nitrate reduction, indole production, glucose fermentation, arginine dihydrolase and urease. None of the substrates embedded in API 20NE and API ID 32GN are assimilated. With API ZYM, positive activities for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, cystine arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase, but negative for lipase (C14), valine arylamidase, trypsin, a-chymotrypsin, a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase. Sensitive to amikacin, ampicillin, cefotaxime, ciprofloxacin, chloramphenicol, erythromycin, gentamicin, imipenem, kanamycin, nalidixic acid, netilmicin, penicillin, rifampicin, streptomycin, tetracycline and vancomycin, weakly resistant to polymyxin, but resistant to cephalothin. The major fatty acids are summed feature 8 (C18 : 1v7c and/or C18 : 1v6c), C16 : 0, C16 : 1v5c, summed feature 2 (iso-C16 : 1 I and/or C14 : 0 3-OH) and C12 : 0. The predominant quinone is ubiquinone Q-8. The main polar 1221
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lipids are phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The type strain KIS83-12T (5KACC 16967T5DSM 27281T) was isolated from soil from Gaui island in the Taean region of South Korea. The DNA G+C content of the type strain is 67.9 mol%.
Acknowledgements
a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol 62, 716– 721. Kluge, A. G. & Farris, J. S. (1969). Quantitative phyletics and the
evolution of anurans. Syst Biol 18, 1–32. Losey, N. A., Stevenson, B. S., Verbarg, S., Rudd, S., Moore, E. R. B. & Lawson, P. A. (2013). Fontimonas thermophila gen. nov., sp. nov., a
moderately thermophilic bacterium isolated from a freshwater hot spring, and proposal of Solimonadaceae fam. nov. to replace Sinobacteraceae Zhou et al. 2008. Int J Syst Evol Microbiol 63, 254– 259.
This study was carried out with the support of the Research Program for Agricultural Science & Technology Development, National Academy of Agricultural Science, Rural Development Administration, Republic of Korea (project no. PJ008666).
Minnikin, D. E., O’Donnell, A. G., Goodfellow, M., Alderson, G., Athalye, M., Schaal, A. & Parlett, J. H. (1984). An integrated
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International Journal of Systematic and Evolutionary Microbiology 64
DOI 10.1099/ijs.0.055574-0
Solimonas terrae sp. nov., isolated from soil Soo-Jin Kim,1 Ji-Young Moon,1 Hang-Yeon Weon,1 Jae-Hyung Ahn,1 Wen-Ming Chen2 and Soon-Wo Kwon1 Correspondence Soon-Wo Kwon
1
[email protected]
2
Agricultural Microbiology Division, National Academy of Agricultural Science, Rural Development Administration, Suwon 441-707, Republic of Korea Laboratory of Microbiology, Department of Seafood Science, National Kaohsiung Marine University, No. 142, Hai-Chuan Rd, Nan-Tzu, Kaohsiung City 811, Taiwan, ROC
A Gram-stain-negative, aerobic, motile, rod-shaped bacterium, designated strain KIS83-12T, was isolated from soil of Gaui island in the Taean region of South Korea. The strain grew at 15–33 6C (optimum, 28 6C), at pH 5.0–8.0 (optimum, pH 7.0). Growth did not occur in the presence of NaCl. The strain was catalase-negative and oxidase-positive. Phylogenetic analysis based on 16S rRNA gene sequences showed that KIS83-12T was most closely related to Solimonas soli DCY12T (96.9 %), Solimonas variicoloris MN28T (96.5 %), Solimonas flava CW-KD 4T (96.5 %) and Solimonas aquatica NAA16T (96.0 %), and formed a robust phyletic lineage with members of the genus Solimonas. The main isoprenoid quinone was Q-8. Major polar lipids included phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. Fatty acids present in large and moderate amounts (.5.0 %) were summed feature 8 (C18 : 1v7c and/or C18 : 1v6c), C16 : 0, C16 : 1v5c, summed feature 2 (iso-C16 : 1 I and/or C14 : 0 3-OH) and C12 : 0. The DNA G+C content was 67.9 mol%. On the basis of the taxonomic data obtained in this study, KIS83-12T represents a novel species of the genus Solimonas, for which the name Solimonas terrae sp. nov. is proposed, with KIS83-12T (5KACC 16967T5DSM 27281T) as the type strain.
The family Solimonadaceae consists of two genera, Solimonas and Fontimonas (Losey et al., 2013). The genus Solimonas was proposed by Kim et al. (2007) for Gramnegative, non-motile, rod-shaped, oxidase-negative and catalase-positive bacteria isolated from soil in a ginseng field. Later, three more species were added to the genus Solimonas. Isolates from freshwater spring were named Solimonas aquatica, Sinobacter flavus and Singularimonas variicoloris and were reclassified as Solimonas flava and Solimonas variicoloris, respectively (Sheu et al., 2011). The genus Solimonas was emended as Gram-negative, rod-shaped, aerobic or facultatively anaerobic, oxidase- and catalasepositive, and motile or non-motile bacteria. Members of the genus Solimonas are characterized by having C16 : 0 and C18 : 1v7c as their major fatty acids, and phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and an uncharacterized aminophospholipid as predominant polar lipids. The main respiratory isoprenoid quinone is Q-8. Bacterial strain KIS83-12T was isolated from the soil of Gaui island in the Taean region of South Korea, using the standard dilution plating technique. Isolation was achieved using a R2A (Difco), and the isolate was routinely cultured The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain KIS83-12T is KF112046. Two supplementary figures are available with the online version of this paper.
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on R2A (Difco) agar at 28 uC and maintained as 15 % (v/v) glycerol suspensions at 280 uC. The 16S rRNA gene of strain KIS83-12T was amplified using the universal primers 9F and 1512R (Weisburg et al., 1991), and was sequenced by Genotec, Daejeon, South Korea. Its sequence was 1433 bp and was compared with other sequences in the EzTaxon-e server (http://eztaxon-e. ezbiocloud.net; Kim et al., 2012). The 16S rRNA gene sequences of strain KIS83-12T and associated species were aligned using the integrated SINA alignment tool from the ARB-SILVA website (Pruesse et al., 2007). Phylogenetic trees based on aligned sequences and evolutionary analyses were reconstructed using MEGA version 5 (Tamura et al., 2011) with three algorithms: neighbour-joining (Saitou & Nei, 1987), maximum-parsimony (Kluge & Farris, 1969) and maximum-likelihood (Felsenstein, 1981) based on 1000 randomly chosen bootstrap replications. Strain KIS83-12T showed highest sequence similarities to the species of the genus Solimonas, Solimonas soli DCY12T (96.9 %), Solimonas variicoloris MN28T (96.5 %), Solimonas flava CW-KD 4T (96.5 %) and Solimonas aquatica NAA16T (96.0 %). Its degree of sequence similarity with other species with validly published names was less than 94.0 %. The neighbourjoining phylogenetic tree revealed that strain KIS83-12T clustered with members of the genus Solimonas; this was supported by the maximum-likelihood and maximumparsimony trees (Fig. 1). 055574 G 2014 IUMS Printed in Great Britain
Solimonas terrae sp. nov.
0.02
97 85
95
Solimonas variicoloris MN 28T (AJ555478) Solimonas flava CW-KD 4T (EF154515) Solimonas soli DCY12T (EF067861)
100
Solimonas terrae KIS83-12T (KF112046)
100
Solimonas aquatica NAA16T (EU303271) Fontimonas thermophila HA-01T (JN415769)
100
Alkanibacter difficilis MN154.3T (AJ313020) Nevskia soli GR15-1T (EF178286)
100
100
Nevskia ramosa Soe1T (AJ001010) Hydrocarboniphaga effusa AP103T (AY363245) 99
100
Hydrocarboniphaga daqingensis B2-9T (EU313812) Rhodanobacter ginsengisoli GR17-7T (EF166075) Dyella japonica XD53T (AB110498)
Arenimonas malthae CC-JY-1T (DQ239766)
99
Thermomonas haemolytica A50-7-3T (AJ300185)
93
Luteimonas mephitis B1953/27.1T (AJ012228)
98
Lysobacter soli DCY21T (EF623862)
87 97
Xanthomonas melonis LMG 8670T (Y10756) Pseudoxanthomonas yeongjuensis GR12-1T (DQ438977) Escherichia coli DSM 30083T (X80725)
Fig. 1. Neighbour-joining phylogenetic tree showing the position of strain KIS83-12T, based on an alignment of 16S rRNA gene sequences. Filled circles indicate that the corresponding branches were also recovered both in the maximum-parsimony and maximum-likelihood trees. Bootstrap percentages (based on 1000 replications) .70 % are shown at branching points. Bar, 0.02 substitutions per nucleotide position.
Strain KIS83-12T was tested for a range of phenotypic, physiological and biochemical characteristics together with type strains of species of the genus Solimonas. The morphology of cells grown on R2A agar at 28 uC for 2 days, after negative staining with 1 % (w/v) sodium phosphotungstic acid (pH 7.0) was investigated with an electron microscope (LEO model 912AB). Gram staining was determined using heat-fixed liquid cultures and a Difco Gram-staining kit, according to the manufacturer’s instructions. Physiological and biochemical properties were tested with API 20NE, API ID 32GN and API ZYM test kits (bioMe´rieux). The test procedure followed the manufacturer’s instructions except that assimilation results were checked after an incubation period of up to 14 days. Catalase activity was assessed by bubble production in 3 % (v/v) H2O2 and oxidase activity was determined using 1 % (w/v) tetramethyl-p-phenylenediamine. Growth at 4, 10, 15, 20, 25, 28, 30, 33, 35, 37 and 40 uC was assessed after 14 days on R2A agar. Growth at pH 4.0–10.0 (at intervals of 1.0 pH unit) was assessed after 14 days in R2A broth; pH was adjusted with citrate/phosphate buffer, Tris/HCl buffer, HCl or NaOH (Breznak & Costilow, 1994). Growth in 0, 0.5, 1, 2 and 3 % (w/v) NaCl was assessed after 14 days in R2A broth. Anaerobic growth was investigated using incubation in the BBL GasPak Anaerobic System (Difco) for 7 days at 28 uC on R2A agar supplemented with 0.5 % (w/v) Na2SO4, 0.5 % (w/v) NaNO3, 0.5 % (w/v) NaHCO3, http://ijs.sgmjournals.org
or 0.02 % (w/v) FeCl3. Casein, starch and tyrosine degradation was examined on R2A plates containing milk powder (5 %, w/v), starch (1 %, w/v) or tyrosine (0.1 %, w/v), respectively. The degradation of CM-cellulose and Tween 80 was examined using R2A supplemented with 1 % (w/v) substrates. DNase activity was determined with DNase test agar (Difco). For antibiotic testing, antimicrobial compound discs (BD) were used as follows: amikacin (30 mg), ampicillin (10 mg), cefotaxime (30 mg), cephalothin (30 mg), chloramphenicol (30 mg), ciprofloxacin (5 mg), erythromycin (15 mg), gentamicin (10 mg), imipenem (10 mg), kanamycin (30 mg), nalidixic acid (30 mg), netilmicin (30 mg), penicillin (10 mg), polymyxin (300 IU), rifampicin (5 mg), streptomycin (10 mg), tetracycline (30 mg) and vancomycin (30 mg). The effect of antimicrobial compounds on cell growth was assessed after 4 days at 30 uC. The diameter of the antibiotic discs was 8 mm. A strain was considered susceptible when the diameter of the inhibition zone was .13 mm, intermediate at 10–12 mm and resistant at ,10 mm. Strain KIS83-12T was aerobic, Gram-stain-negative, rod-shaped, non-spore-forming and motile by means of a single polar flagellum (Fig. S1 available in the online Supplementary Material). Growth occurred at 15–33 uC (optimum, 28 uC), at pH 5.0–8.0 (optimum, pH 7.0). Growth did not occur in the presence of NaCl. Strain KIS83-12T grew on R2A, weakly on nutrient agar (Difco), but not on trypticase soy agar (Difco) or McConkey 1219
S.-J. Kim and others
Table 1. Phenotypic comparison of strain KIS83-12T and species of the genus Solimonas Strain: 1, KIS83-12T; 2, Solimonas aquatica NAA 16T; 3, Solimonas flava CW-KD 4T; 4, Solimonas soli DCY 12T; 5, Solimonas variicoloris MN 28T. All strains are positive for aesculin hydrolysis and oxidase, but negative for nitrate reduction, indole production, glucose fermentation and arginine dihydrolase. None of the strains assimilate D-mannose, potassium gluconate, trisodium citrate or phenylacetic acid. All strains showed positive activities for esterase lipase (C8), acid phosphatase and naphthol-AS-BI-phosphohydrolase, but negative activities for lipase (C14), trypsin, achymotrypsin, a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase. +, Positive; 2, negative; W, weakly positive. Characteristic Isolation source Colony colour Cell morphology Cell size (mm) Motility Catalase Temperature range (uC) Growth on 1 % NaCl (w/v) DNA hydrolysis Urease Gelatin hydrolysis b-Galactosidase Assimilation of: D-Glucose L-Arabinose D-Mannitol N-Acetylglucosamine Maltose Capric acid Adipic acid Malic acid Enzymic activity of: Alkaline phosphatase Esterase (C4) Leucine arylamidase Valine arylamidase Cystine arylamidase b-Glucosidase Antibiotic susceptibility to: Ampicillin (10 mg) Cephalothin (30 mg) Imipenem (10 mg) Penicillin (10 mg) Polymyxin (300 IU) DNA G+C content (mol%)
1
2
3
4
Water * White Short rod 0.6–0.861.0–1.6 +a – 20–40a +a –a + – –
Soil Yellow Rod 0.5–0.661.3–1.8 –a + 15–42a +a –a – + –
Soil Light yellow Rod 0.5–0.661.3–1.9 +a + 20–42a +a +a – – –
Biofilterd Yellow Long rod 0.4–0.561.6–3.1 –a + 20–42a +a –a – + –
– – – – – – – –
+ + + + + – + –
+ – + – – + – +
– – – – – – – –
– – – – – – – –
+ + + – + –
– – + + – –
+ + + – – –
+ + – – – –
+ + – – – +
+ – + +
+ + – – – 66.2a
– – + – + 65.1b
+ – + +
+ + + + + 64.9d
W
b
5
Soil Light yellow Long rod 0.5–0.661.5–2.4 + – 15–33 – – – + +
67.9
a
c
W
68.4a
*Data from: a, Sheu et al. (2011); b, Zhou et al., (2008); c, Kim et al., (2007); d, Friedrich & Lipski, (2008).
For fatty acid analysis, strain KIS83-12T and the four reference strains were grown on R2A at 28 uC for 48 h to the
late exponential phase. The fatty acid methyl esters were obtained by saponification, methylation and extraction, and analysed using the Sherlock Microbial Identification System (MIDI) according to the manufacturer’s instructions. Fatty acid methyl esters were analysed by using the Microbial Identification software package (Sherlock Version 6.1; MIDI database: TSBA6). Polar lipids were examined using twodimensional TLC. The reagents used for detection of polar lipids were as follows: 5 % (v/v) ethanolic molybdophosphoric acid was used for detection of all lipids, ninhydrin
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International Journal of Systematic and Evolutionary Microbiology 64
Agar (Difco). Table 1 shows differential characteristics of KIS83-12T and other strains of species of the genus Solimonas. Strain KIS83-12T could be differentiated from species of the genus Solimonas with validly published names on the basis of morphology, catalase activity, temperature range for growth, NaCl tolerance, ability to hydrolyse several substrates, assimilation patterns and sensitivity to several antibiotics.
Solimonas terrae sp. nov.
reagent for lipids containing free amino groups, Zinzadze reagent for phosphorus-containing lipids and a-naphthol reagent for glycolipids (Collins et al., 1980). Quinones were characterized as described by Minnikin et al. (1984). The G+C content was determined by the fluorometric method (Gonzalez & Saiz-Jimenez, 2002) using SYBR Green 1 and a real-time PCR thermocycler (Bio-Rad). Strain KIS83-12T contained large and moderate amounts of the fatty acids summed feature 8 (C18 : 1v7c and/or C18 : 1v6c; 39.8 %), C16 : 0 (15.6 %), C16 : 1v5c (10.3 %), summed feature 2 (isoC16 : 1 I and/or C14 : 0 3-OH; 5.6 %) and C12 : 0 (5.0 %), along with smaller amounts of several fatty acids (Table 2). These fatty acids present in strain KIS83-12T in large and moderate amounts were detected in other species of the genus
Table 2. Cellular fatty acid compositions (%) of strain KIS8312T and the type strains of species of the genus Solimonas Strain: 1, KIS83-12T; 2, Solimonas aquatica NAA 16T; 3, Solimonas flava CW-KD 4T; 4, Solimonas soli DCY 12T; 5, Solimonas variicoloris MN 28T. All fatty acid data were obtained in this study from cells grown on R2A for 48 h. –, not detected or ,0.5% of the total fatty acids. Fatty acid C10 : 0 C12 : 0 iso-C12 : 0 C12 : 0 2-OH C12 : 0 3-OH C14 : 0 iso-C14 : 0 3-OH C14 : 0 2-OH C16 : 0 iso-C16 : 0 C16 : 0 2-OH C16 : 0 3-OH C16 : 1v5c C16 : 1v11c C16 : 1v7c alcohol C17 : 0 C17 : 1v6c C17 : 0 cyclo 10-Methyl C17 : 0 C18 : 0 iso-C18 : 1 H C19 : 0 cyclo v8c Summed feature* 1 2 3 8
1
2
3
4
5
2.2 5.0 – – 0.8 4.7 – 1.4 15.6 3.1 – – 10.3 2.5 0.6 – – – 1.8 – 0.7 –
– 6.9 0.7 – – 4.3 – – 18.2 5.2 – 0.9 6.5 – – – – 0.7 – – 0.7 –
– 3.7 1.2 2.2 1.2 5.5 1.1 – 13.3 7.3 – – 6.2 5.4 – – – – – – 1.2 –
– 4.4 – 3.0 – 5.2 – – 11.5 – 0.8 – 12.1 – – 1.4 0.8 – – – – 2.0
– 5.5 0.6 1.6 – 5.4 – – 15.2 2.2 – – 3.7 4.1 – – – – – 0.7 – –
– 5.6 3.6 39.8
– 6.9 23.3 23.8
– 7.9 3.3 37.3
1.3 9.4 4.0 41.3
– 9.1 3.7 45.8
*Summed features are combinations of fatty acids that cannot be separated by the MIDI system. Summed feature 1 comprises C13 : 0 3OH and/or iso-C15 : 1 H; summed feature 2 comprises iso-C16 : 1 I and/ or C14 : 0 3-OH; summed feature 3 comprises C16 : 1v7c and/or C16 : 1v6c; summed feature 8 comprises C18 : 1v7c and/or C18 : 1v6c. http://ijs.sgmjournals.org
Solimonas, despite small quantitative differences. Strain KIS83-12T contained large amounts of phosphatidylethanolamine (PE), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG) as the major polar lipids and it also contained a small amount of aminophospholipid and small amounts of several aminolipids (Fig. S2). The polar lipid profiles of other strains of species of the genus Solimonas indicated that aminophospholipids along with PE, PG and DPG were major polar lipids (Sheu et al., 2011). However, strain KIS83-12T contained only a small amount of aminophospholipid. Strain KIS83-12T contained Q-8 as the predominant respiratory quinone, which is in accordance with the genus Solimonas (Sheu et al., 2011). The genomic DNA G+C content of strain KIS83-12T was 67.9 mol%. It is evident, based on a 16S rRNA gene sequence similarity of less than 97 % (,96.9 %) with species with validly published names, a phylogenetic clustering with other species of the genus Solimonas (Fig. 1) and differential taxonomic characteristics (Table 1), that strain KIS83-12T should be classified as a novel species, distinct from currently recognized species of the genus Solimonas, and the name Solimonas terrae sp. nov. is proposed. Description of Solimonas terrae sp. nov. Solimonas terrae (ter9rae. L. gen. n. terrae of the soil). Cells are aerobic, Gram-stain-negative, motile rods, 0.5– 0.6 mm61.5–2.4 mm. Catalase-negative and oxidase-positive. Grows on R2A, weakly on nutrient agar, but not on trypticase soy agar or McConkey agar. Colonies are light yellow, convex, round with entire edges on R2A agar at 28 uC. Grows in the temperature range of 15–33 uC (optimum, 28 uC) and at pH5.0–8.0 (optimum 7.0). Grows without NaCl. Hydrolyses tyrosine, but not casein, cellulose, chitin, DNA, hypoxanthine, Tween 80, starch or xanthine. With API 20NE, positive for aesculin hydrolysis, gelatin hydrolysis and b-galactosidase (PNG), but negative for nitrate reduction, indole production, glucose fermentation, arginine dihydrolase and urease. None of the substrates embedded in API 20NE and API ID 32GN are assimilated. With API ZYM, positive activities for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, cystine arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase, but negative for lipase (C14), valine arylamidase, trypsin, a-chymotrypsin, a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase. Sensitive to amikacin, ampicillin, cefotaxime, ciprofloxacin, chloramphenicol, erythromycin, gentamicin, imipenem, kanamycin, nalidixic acid, netilmicin, penicillin, rifampicin, streptomycin, tetracycline and vancomycin, weakly resistant to polymyxin, but resistant to cephalothin. The major fatty acids are summed feature 8 (C18 : 1v7c and/or C18 : 1v6c), C16 : 0, C16 : 1v5c, summed feature 2 (iso-C16 : 1 I and/or C14 : 0 3-OH) and C12 : 0. The predominant quinone is ubiquinone Q-8. The main polar 1221
S.-J. Kim and others
lipids are phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The type strain KIS83-12T (5KACC 16967T5DSM 27281T) was isolated from soil from Gaui island in the Taean region of South Korea. The DNA G+C content of the type strain is 67.9 mol%.
Acknowledgements
a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol 62, 716– 721. Kluge, A. G. & Farris, J. S. (1969). Quantitative phyletics and the
evolution of anurans. Syst Biol 18, 1–32. Losey, N. A., Stevenson, B. S., Verbarg, S., Rudd, S., Moore, E. R. B. & Lawson, P. A. (2013). Fontimonas thermophila gen. nov., sp. nov., a
moderately thermophilic bacterium isolated from a freshwater hot spring, and proposal of Solimonadaceae fam. nov. to replace Sinobacteraceae Zhou et al. 2008. Int J Syst Evol Microbiol 63, 254– 259.
This study was carried out with the support of the Research Program for Agricultural Science & Technology Development, National Academy of Agricultural Science, Rural Development Administration, Republic of Korea (project no. PJ008666).
Minnikin, D. E., O’Donnell, A. G., Goodfellow, M., Alderson, G., Athalye, M., Schaal, A. & Parlett, J. H. (1984). An integrated
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