International Journal of Systematic and Evolutionary Microbiology (2014), 64, 1024–1029
DOI 10.1099/ijs.0.053827-0
Roseomonas soli sp. nov., isolated from an agricultural soil cultivated with Chinese cabbage (Brassica campestris) Dong-Uk Kim and Jong-Ok Ka Correspondence Jong-Ok Ka [email protected]
Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-742, Republic of Korea A bacterial strain, designated 5N26T, was isolated from an agricultural soil cultivated with Chinese cabbage (Brassica campestris). Cells of this strain were Gram-reaction-negative, strictly aerobic, motile, non-spore-forming rods, and catalase- and urease-negative. The major fatty acids of strain 5N26T were C16 : 0 (7.5 %), C18 : 1 2-OH (13.4 %) and summed feature 8 (C18 : 1v6c and/or C18 : 1v7c; 63.2 %). The polar lipid profile contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, phosphatidylmonomethylethanolamine and one unidentified aminolipid. The G+C content of the genomic DNA of strain 5N26T was 68.3 mol%. 16S rRNA gene sequence analysis showed that strain 5N26T was phylogenetically related to Roseomonas lacus TH-G33T and Roseomonas terrae DS-48T (97.0 % and 96.6 % sequence similarity, respectively). The results of genotypic and phenotypic data showed that strain 5N26T could be distinguished from phylogenetically related species, and that this strain represented a novel species within the genus Roseomonas, for which the name Roseomonas soli sp. nov. (type strain 5N26T5KACC 16376T5NBRC 109097T) is proposed.
The genus Roseomonas was proposed by Rihs et al. (1993) on the basis of the classification of an alphaproteobacterial genus with typical pink pigmentation, coccoid–rod shape and oxidative metabolism. Since the description of three novel species in 1993, the number of novel species with validly published names has increased. Species of the genus Roseomonas have been isolated frequently from blood, wounds, exudates, abscesses and genito-urinary specimens (Bibashi et al., 2000; Rihs et al., 1993; Sandoe et al., 1997; Subudhi et al., 2001). Species of the genus Roseomonas may occur widely in nature and recently representatives were isolated from water-related environmental sources such as freshwater sediment (Jiang et al., 2006), water distribution systems (Gallego et al., 2006), deep-water marine invertebrates (Sfanos et al., 2005) and pond water (Furuhata et al., 2008). Phylogenetic analysis based on 16S rRNA gene sequences indicated that species of the genus Roseomonas were frequently included in various bacterial communities (Islam et al., 2012; Tamaki et al., 2005). At the time of writing, the genus consisted of 16 species with validly published names: Roseomonas lacus (Jiang et al., 2006), R. terrae (Yoon et al., 2007), R. aquatica (Gallego et al., 2006), R. ludipueritiae (Sa´nchez-Porro et al., 2009), R. rosea (Sa´nchez-Porro et al., 2009), R. mucosa (Bard et al., 2010), R. gilardii (type species, with two subspecies, R. gilardii subsp. gilardii and R. gilardii subsp. rosea) (Han et al., The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain 5N26T is JN575264.1.
1024
2003; Rihs et al., 1993), R. aestuarii (Venkata Ramana et al., 2010), R. cervicalis (Rihs et al., 1993), R. vinacea (Zhang et al., 2008), R. aerophila (Kim et al., 2013), R. stagni (Furuhata et al., 2008), R. pecuniae (Lopes et al., 2011), R. frigidaquae (Kim et al., 2009), R. aerilata (Yoo et al., 2008) and R. riguiloci (Baik et al., 2012). Strain 5N26T was isolated from agricultural soil cultivated with Chinese cabbage using the standard dilution plating technique on R2A agar (Difco). After plating, the plate was incubated at 30 uC for 7 days and a white-pigmented bacterial strain, 5N26T, was isolated. The growth of strain 5N26T was assessed on nutrient agar (NA; Difco), R2A agar (Difco), trypticase soy agar (TSA; Difco), MacConkey agar (Difco) and BCYE agar (BBL). The methods of Smibert & Krieg (1994) were used for determination of oxidase and catalase activities, and hydrolysis of casein, hypoxanthine, starch, DNA, xanthine, Tween 80, tyrosine (0.5 %, w/v), pectin (0.5 %, w/v), CMcellulose (0.1 %; Sigma) and chitin (1 %, w/v). In addition, aesculin hydrolysis was assessed using the API 20NE kit (bioMe´rieux) at 30 uC. Phase-contrast microscopy (AXIO; Zeiss) was used for cell motility and morphology studies with cells grown for 5 days at 30 uC on R2A. The temperature range, tolerance of NaCl and pH range for growth were examined on R2A agar after cultivation for 5 days at 30 uC. Tolerance of NaCl was tested on 0–10 % (w/v) NaCl (at 0.5 % intervals) R2A agar prepared according to the formula of the Difco medium. The temperature range for 053827 G 2014 IUMS Printed in Great Britain
Roseomonas soli sp. nov.
growth was tested on R2A agar (Difco) at 5, 10, 15, 20, 35, 37, 40 and 45 uC. The pH range for growth was determined on R2A agar (Difco) adjusted to pH 2–10 at 1 pH unit intervals. Other physiological and biochemical properties were tested using the commercial API ZYM, API 20E and API 20NE systems (bioMe´rieux). The API ZYM tests were read after 4 h of incubation at 37 uC, and the other API tests after 48 h at 30 uC. All kits were used according to the manufacturer’s instructions. For analysis of the cellular fatty acids, strain 5N26T and those representing other related species were grown concurrently on R2A agar for 2 days at 30 uC in our laboratory. The cellular fatty acids were extracted, methylated and separated by gas chromatography (model 6890; Hewlett Packard) according to the protocol of the Sherlock Microbial Identification System (MIDI, 1999). The fatty acid methyl esters were identified and quantified using the TSBA 6 database (version 6.10) of the Sherlock Microbial Identification System (MIDI). Polar lipids were analysed by two-dimensional TLC. The DNA G+C content (mol%) was measured by HPLC analysis of the deoxyribonucleotides using a reversed-phase column (Supelcosil LC-18 S; Supelco) (Mesbah et al., 1989). The identification of isoprenoid quinones followed a previously described HPLC method (Groth et al., 1996). Chromosomal DNA was isolated from the strain 5N26T with the Wizard Genomic DNA Purification kit (Promega). The 16S rRNA gene was amplified by PCR using the universal primers 27f and 1492r (Baker et al., 2003). Sequencing was performed with an ABI Prism BigDye Terminator Cycle
Sequencing Ready kit (Applied Biosystems) according to the manufacturer’s instructions with the sequencing primers 519r, 926f (Lane, 1991) and 1055r (Lee et al., 1993). Partial 16S rRNA gene sequences were assembled using SEQMAN software (DNASTAR). Similarity of the almost full-length sequence to those of related taxa was determined using the EzTaxon server (Chun et al., 2007) and sequences from 5N26T and related taxa (retrieved from the NCBI database) were aligned using the multiple sequence alignment program CLUSTAL W (Thompson et al., 1994). Phylogenetic trees were inferred using neighbour-joining (Saitou & Nei, 1987) and maximum-parsimony methods (Fitch, 1971) by using the MEGA4 software program (Tamura et al., 2007) with bootstrap values based on 1000 replications (Felsenstein, 1985). Strain 5N26T grew on NA, R2A agar, TSA and BCYE agar, but not on MacConkey agar. The strain showed poor tolerance of NaCl and a narrower pH range for growth than R. lacus KACC 11678T and R. terrae KACC 12677T. However, strain 5N26T had a broader range of growth temperature than these two species. It showed white-coloured colonies while R. lacus KACC 11678T and R. terrae KACC 12677T had yellow and reddish pink colonies, respectively. In addition, the novel isolate showed negative reactions for catalase and urease, while its closest relatives exhibited positive reactions for these enzymes. The detailed morphological, physiological and biochemical characteristics of strain 5N26T and other species of the genus Roseomonas are shown in Table 1. Strain 5N26T had three major fatty acids, C16 : 0 (7.5 %), C18 : 1 2-OH (13.4 %) and summed feature 8 (C18 : 1v6c
Table 1. Differential phenotypic characteristics of strain 5N26T and type strains of closely related species Strains: 1, 5N26T (Roseomonas soli sp. nov.); 2, R. lacus KACC 11678T; 3, R. terrae KACC 12677T; 4, R. gilardii KACC 11652T; 5, R. cervicalis KACC 11686T; 6, R. ludipueritiae KACC 13843T. All data were obtained from this study. All strains are positive for aerobic growth and for alkaline phosphatase, esterase (C4), esterase lipase (C8), acid phosphatase and naphthol-AS-BI-phosphohydrolase activities, but negative for growth on MacConkey agar, hydrolysis of starch, CM-cellulose, Tween 80, chitin, hypoxanthine, xanthine and aesculin, valine arylamidase, cystine arylamidase, trypsin, lipase (C14), a-chymotrypsin, b-glucuronidase, a-galactosidase, b-galactosidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase, a-fucosidase, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase and tryptophan deaminase activities, H2S production, fermentation/oxidation of D-glucose, D-mannitol, inositol, D-sorbitol, L-rhamnose, sucrose, melibiose, amygdalin and glucose, and assimilation of D-mannose, N-acetylglucosamine, maltose, gluconate, caprate, adipate, malate, citrate and phenylacetate. +, Positive; (+), weakly positive; 2, negative. Characteristic Habitat Temperature range for growth (uC) Colony colour Catalase activity Hydrolysis of: Urea Casein Gelatin Assimilation of: D-Arabinose L-Arabinose
http://ijs.sgmjournals.org
1
2
3
4
5
6
Cabbage rhizosphere 15–40 White 2
Soil
Soil 4–30 Reddish pink +
Flood-plain sediment 4–30 Reddish pink +
Soil
10–30 Yellow +
Cabbage rhizosphere 4–35 Pinkish yellow +
4–30 Reddish pink +
2 2 2
+ 2 2
+ + +
+ + +
+ + +
+ + +
(+) 2
2 +
2 2
2 +
2 +
2 2
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D.-U. Kim and J.-O. Ka
Table 2. Cellular fatty acid contents of strain 5N26T and related species Strains: 1, 5N26T (Roseomonas soli sp. nov.); 2, R. lacus KACC 11678T; 3, R. terrae KACC 12677T; 4, R. gilardii KACC 11652T; 5, R. cervicalis KACC 11686T; 6, R. ludipueritiae KACC 13843T. All data were obtained from this study. Prior to fatty acid extraction, all strains were grown on R2A agar (Difco) at 30 uC for 2 days. Values are percentages of total fatty acids, and only fatty acids representing more than 1 % for at least one of the strains are shown. –, Not detected; TR, trace amounts (,1 %). Fatty acid C12 : 0 C14 : 0 C14 : 0 2-OH C16 : 0 C16 : 0 2-OH C16 : 0 3-OH C16 : 1v5c C18 : 0 C18 : 1 2-OH 11-Methyl C18 : 1v7c C19 : 0 cyclo v7c Summed feature 3* Summed feature 4* Summed feature 8*
1
2
–
– – – 4.6 –
TR
– 7.5 – 1.6 2.4 – 13.4 – 4.4 4.1 1.3 63.2
TR
3.3
3
4
–
–
TR
TR
– 9.4 1.7 1.3 2.6
TR
TR
7.6 4.8 2.2 4.3 – 70.3
12.3
– 25.0 TR
1.1 – 1.1 1.7
TR
TR
11.2 1.1 – 57.5
35.8 1.1 – 31.0
5
6
1.0 – 1.1 2.2 – 2.4 15.3 14.3 – TR 1.1 1.5 2.6 2.4 – – 3.1 2.3 – – 16.4 23.2 3.1 4.9 – – 55.4 43.6
*Summed features are groups of two or three fatty acids that cannot be separated using the MIDI system. Summed feature 3 comprises C16 : 1v7c and/or C16 : 1v6c; summed feature 4 comprises iso-C17 : 1 I and/or anteisoC17 : 1 B and summed feature 8 comprises C18 : 1v6c and/or C18 : 1v7c.
and/or C18 : 1v7c; 63.2 %), which are frequently found in species of the genus Roseomonas. The strain also had summed feature 4 (iso-C17 : 1 I and/or anteiso B) which was not found in other species of the genus Roseomonas. The
(a)
differential fatty acid characteristics of strain 5N26T and other species of the genus Roseomonas are shown in Table 2. The polar lipids present in 5N26T were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine (PE), phosphatidylcholine, phosphatidylmonomethylethanolamine and one unidentified aminolipid (Fig. 1). Previously, phosphatidylmonomethylethanolamine was detected in the polar lipid profiles of other species of the genus Roseomonas such as R. lacus CIP 109168T, R. mucosa CIP 108268T and R. cervicalis CIP 104027T (Sa´nchez-Porro et al., 2009). When the PE spot in the molybdenum blue image is compared with the corresponding spot in the ninhydrin image, the former is smaller than the latter. Another aminolipid spot (Fig. 1b, arrow) which stains only with ninhydrin and not with molybdenum blue might be present at the lower right part of the PE spot. But due to the high similarity in its chromatographic motility to that of PE it cannot be separated from PE. The presence of the second aminolipid has been reported in species of the genus Roseomonas (Sa´nchez-Porro et al., 2009). The polar lipid data showed that strain 5N26T belonged to the genus Roseomonas and additionally it appeared to have two other aminolipids including phosphatidylmonomethylethanolamine in its polar lipid pattern, making it novel among species of the genus Roseomonas. The DNA G+C content of strain 5N26T was 68.3 mol% and ubiquinone 10 (Q-10) was the predominant isoprenoid quinone. The sequence analyses of the 16S rRNA gene showed that strain 5N26T was closely related to R. lacus TH-G33T and R. terrae DS-48T. A previously reported 1376 bp 16S rRNA gene sequence of R. lacus TH-G33T had 41 nt differences from 5N26T. R. terrae DS-48T had 47 nt differences in 1414 bp of 16S rRNA gene sequence. The 16S rRNA gene sequence similarities of 5N26T were 97.0 % and 96.6 % with R. lacus TH-G33 T and R. terrae DS-48T, respectively. The neighbour-joining tree based on 16S rRNA gene sequences showed that strain 5N26T was grouped with the
(b) DPG PME
PME
PE
PE
PG AL PC
(c) DPG
PME PE
PG
AL PC
Fig. 1. Polar lipid profile of strain 5N26T after two-dimensional TLC, detected using molybdatophosphoric acid (a), ninhydrin (b) and molybdenum blue (c). PE, phosphatidylethanolamine; PME, phosphatidylmonomethylethanolamine; DPG, diphosphatidylglycerol; PG, phosphatidylglycerol; PC, phosphatidylcholine; AL, unknown aminolipid. Arrow, aminolipid spot which stains only with ninhydrin and not with molybdenum blue. 1026
International Journal of Systematic and Evolutionary Microbiology 64
Roseomonas soli sp. nov.
0.02
99
Roseomonas aestuarii JC17T (FM244739)
67
Roseomonas cervicalis ATCC 49957T (AY150047)
53
Roseomonas ludipueritiae 170/96T (AJ488504) Roseomonas aquatica TR53T (AM231587) Roseomonas rosea 173/96T (AJ488505)
77
Roseomonas pecuniae N75T (GU168019)
90
Roseomonas vinacea CPCC 100056T (EF368368) 99
59
Roseomonas aerilata 5420S-30T (EF661571)
T 72 Roseomonas gilardii subsp. rosea ATCC BAA-691 (AY220740) 100 Roseomonas gilardii subsp. gilardii ATCC 49956T (AY150045)
Roseomonas mucosa ATCC BAA-692T (AF538712) 52
Roseomonas riguiloci 03SU10-PT (HQ436503) Roseomonas stagni HS-69T (AB369258)
99 65
Roseomonas frigidaquae CW67T (EU290160) Roseomonas soli 5N26T (JN575264)
100
Roseomonas lacus TH-G33T (AJ786000) 89
Roseomonas terrae DS-48T (EF363716) Roseomonas fauriae ATCC 49958T (AY150046)
Fig. 2. Phylogenetic dendrogram reconstructed from a comparative analysis of 16S rRNA gene sequences showing the relationship between strain 5N26T and related species. Bootstrap values (expressed as percentages of 1000 replications) greater than 50 % are shown at branch points. Filled circles indicate that the corresponding branches were also recovered in the maximum-parsimony tree. Bar, 0.02 substitutions per nucleotide position.
members of the genus Roseomonas and formed a distinct phylogenetic line distinguishable from other species of the genus Roseomonas (Fig. 2). In conclusion, the main characteristics of strain 5N26T were in accordance with the description of the genus Roseomonas, but the new isolate could be distinguished from the other species of the genus Roseomonas by a combination of physiological and biochemical features and minor differences in their fatty acid profiles. Therefore, on the basis of the data from the polyphasic study presented here, strain 5N26T represents a novel species in the genus Roseomonas, for which the name Roseomonas soli sp. nov. is proposed. Description of Roseomonas soli sp. nov. Roseomonas soli (so9li. L. gen. n. soli of soil, the source of the type strain). Cells are Gram-reaction-negative, strictly aerobic, motile, non-spore-forming rods, 0.7–1.0 mm in diameter and 1.9– 2.4 mm in length after cultivation for 5 days at 30 uC on R2A agar. Colonies are 0.4–1.0 mm in diameter, smooth, circular, convex and white in colour after cultivation for 5 days at 30 uC on R2A agar. Grows on LB, NA, R2A agar, TSA and BCYE agar, but not on MacConkey agar. Catalase, urease and oxidase are negative. Growth occurs at 15–40 uC (optimal growth occurs at 30 uC) and pH 5.0–8.5 (optimal growth occurs at pH 6.5), but no growth occurs with 1 % NaCl. Does http://ijs.sgmjournals.org
not hydrolyse casein, gelatin, starch, tyrosine, Tween 80, chitin, CM-cellulose, hypoxanthine, urea, xanthine or aesculin. Positive (in API ZYM strips) for alkaline phosphatase, esterase (C4), esterase lipase (C8), acid phosphatase and naphthol-AS-BI-phosphohydrolase, and negative for leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, lipase (C14), a-chymotrypsin, b-glucuronidase, a-galactosidase, b-galactosidase, a-glucosidase, b-glucosidase, N-acetylb-glucosaminidase, a-mannosidase and a-fucosidase. Acetoin production is positive but negative for arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, citrate utilization, H2S production, tryptophan deaminase, indole production and gelatinase. Fermentation/oxidation of D-glucose, D-mannitol, inositol, D-sorbitol, L-rhamnose, sucrose, melibiose, amygdalin and D-arabinose are negative (in API 20E strips). Acidification of glucose is negative and assimilation of D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetylglucosamine, maltose, gluconate, caprate, adipate, malate, citrate and phenylacetate are negative (in API 20NE strips). Ubiquinone 10 (Q-10) is the predominant quinone. The major fatty acids are C16 : 0, C18 : 1 2-OH and summed feature 8 (C18 : 1v6c and/or C18 : 1 v7c). The polar lipids are diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, phosphatidylmonomethylethanolamine and one unidentified aminolipid. The type strain is 5N26T (5KACC 16376T5NBRC 109097T), isolated from rhizosphere soil cultivated with Chinese cabbage (Brassica campestris L.) in Namyangju, 1027
D.-U. Kim and J.-O. Ka
Korea. The DNA G+C content of the type strain is 68.3 mol%.
Acknowledgements This work was supported by a grant from the Regional SubGenBank Support Program of Rural Development Administration, Republic of Korea.
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DOI 10.1099/ijs.0.053827-0
Roseomonas soli sp. nov., isolated from an agricultural soil cultivated with Chinese cabbage (Brassica campestris) Dong-Uk Kim and Jong-Ok Ka Correspondence Jong-Ok Ka [email protected]
Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-742, Republic of Korea A bacterial strain, designated 5N26T, was isolated from an agricultural soil cultivated with Chinese cabbage (Brassica campestris). Cells of this strain were Gram-reaction-negative, strictly aerobic, motile, non-spore-forming rods, and catalase- and urease-negative. The major fatty acids of strain 5N26T were C16 : 0 (7.5 %), C18 : 1 2-OH (13.4 %) and summed feature 8 (C18 : 1v6c and/or C18 : 1v7c; 63.2 %). The polar lipid profile contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, phosphatidylmonomethylethanolamine and one unidentified aminolipid. The G+C content of the genomic DNA of strain 5N26T was 68.3 mol%. 16S rRNA gene sequence analysis showed that strain 5N26T was phylogenetically related to Roseomonas lacus TH-G33T and Roseomonas terrae DS-48T (97.0 % and 96.6 % sequence similarity, respectively). The results of genotypic and phenotypic data showed that strain 5N26T could be distinguished from phylogenetically related species, and that this strain represented a novel species within the genus Roseomonas, for which the name Roseomonas soli sp. nov. (type strain 5N26T5KACC 16376T5NBRC 109097T) is proposed.
The genus Roseomonas was proposed by Rihs et al. (1993) on the basis of the classification of an alphaproteobacterial genus with typical pink pigmentation, coccoid–rod shape and oxidative metabolism. Since the description of three novel species in 1993, the number of novel species with validly published names has increased. Species of the genus Roseomonas have been isolated frequently from blood, wounds, exudates, abscesses and genito-urinary specimens (Bibashi et al., 2000; Rihs et al., 1993; Sandoe et al., 1997; Subudhi et al., 2001). Species of the genus Roseomonas may occur widely in nature and recently representatives were isolated from water-related environmental sources such as freshwater sediment (Jiang et al., 2006), water distribution systems (Gallego et al., 2006), deep-water marine invertebrates (Sfanos et al., 2005) and pond water (Furuhata et al., 2008). Phylogenetic analysis based on 16S rRNA gene sequences indicated that species of the genus Roseomonas were frequently included in various bacterial communities (Islam et al., 2012; Tamaki et al., 2005). At the time of writing, the genus consisted of 16 species with validly published names: Roseomonas lacus (Jiang et al., 2006), R. terrae (Yoon et al., 2007), R. aquatica (Gallego et al., 2006), R. ludipueritiae (Sa´nchez-Porro et al., 2009), R. rosea (Sa´nchez-Porro et al., 2009), R. mucosa (Bard et al., 2010), R. gilardii (type species, with two subspecies, R. gilardii subsp. gilardii and R. gilardii subsp. rosea) (Han et al., The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain 5N26T is JN575264.1.
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2003; Rihs et al., 1993), R. aestuarii (Venkata Ramana et al., 2010), R. cervicalis (Rihs et al., 1993), R. vinacea (Zhang et al., 2008), R. aerophila (Kim et al., 2013), R. stagni (Furuhata et al., 2008), R. pecuniae (Lopes et al., 2011), R. frigidaquae (Kim et al., 2009), R. aerilata (Yoo et al., 2008) and R. riguiloci (Baik et al., 2012). Strain 5N26T was isolated from agricultural soil cultivated with Chinese cabbage using the standard dilution plating technique on R2A agar (Difco). After plating, the plate was incubated at 30 uC for 7 days and a white-pigmented bacterial strain, 5N26T, was isolated. The growth of strain 5N26T was assessed on nutrient agar (NA; Difco), R2A agar (Difco), trypticase soy agar (TSA; Difco), MacConkey agar (Difco) and BCYE agar (BBL). The methods of Smibert & Krieg (1994) were used for determination of oxidase and catalase activities, and hydrolysis of casein, hypoxanthine, starch, DNA, xanthine, Tween 80, tyrosine (0.5 %, w/v), pectin (0.5 %, w/v), CMcellulose (0.1 %; Sigma) and chitin (1 %, w/v). In addition, aesculin hydrolysis was assessed using the API 20NE kit (bioMe´rieux) at 30 uC. Phase-contrast microscopy (AXIO; Zeiss) was used for cell motility and morphology studies with cells grown for 5 days at 30 uC on R2A. The temperature range, tolerance of NaCl and pH range for growth were examined on R2A agar after cultivation for 5 days at 30 uC. Tolerance of NaCl was tested on 0–10 % (w/v) NaCl (at 0.5 % intervals) R2A agar prepared according to the formula of the Difco medium. The temperature range for 053827 G 2014 IUMS Printed in Great Britain
Roseomonas soli sp. nov.
growth was tested on R2A agar (Difco) at 5, 10, 15, 20, 35, 37, 40 and 45 uC. The pH range for growth was determined on R2A agar (Difco) adjusted to pH 2–10 at 1 pH unit intervals. Other physiological and biochemical properties were tested using the commercial API ZYM, API 20E and API 20NE systems (bioMe´rieux). The API ZYM tests were read after 4 h of incubation at 37 uC, and the other API tests after 48 h at 30 uC. All kits were used according to the manufacturer’s instructions. For analysis of the cellular fatty acids, strain 5N26T and those representing other related species were grown concurrently on R2A agar for 2 days at 30 uC in our laboratory. The cellular fatty acids were extracted, methylated and separated by gas chromatography (model 6890; Hewlett Packard) according to the protocol of the Sherlock Microbial Identification System (MIDI, 1999). The fatty acid methyl esters were identified and quantified using the TSBA 6 database (version 6.10) of the Sherlock Microbial Identification System (MIDI). Polar lipids were analysed by two-dimensional TLC. The DNA G+C content (mol%) was measured by HPLC analysis of the deoxyribonucleotides using a reversed-phase column (Supelcosil LC-18 S; Supelco) (Mesbah et al., 1989). The identification of isoprenoid quinones followed a previously described HPLC method (Groth et al., 1996). Chromosomal DNA was isolated from the strain 5N26T with the Wizard Genomic DNA Purification kit (Promega). The 16S rRNA gene was amplified by PCR using the universal primers 27f and 1492r (Baker et al., 2003). Sequencing was performed with an ABI Prism BigDye Terminator Cycle
Sequencing Ready kit (Applied Biosystems) according to the manufacturer’s instructions with the sequencing primers 519r, 926f (Lane, 1991) and 1055r (Lee et al., 1993). Partial 16S rRNA gene sequences were assembled using SEQMAN software (DNASTAR). Similarity of the almost full-length sequence to those of related taxa was determined using the EzTaxon server (Chun et al., 2007) and sequences from 5N26T and related taxa (retrieved from the NCBI database) were aligned using the multiple sequence alignment program CLUSTAL W (Thompson et al., 1994). Phylogenetic trees were inferred using neighbour-joining (Saitou & Nei, 1987) and maximum-parsimony methods (Fitch, 1971) by using the MEGA4 software program (Tamura et al., 2007) with bootstrap values based on 1000 replications (Felsenstein, 1985). Strain 5N26T grew on NA, R2A agar, TSA and BCYE agar, but not on MacConkey agar. The strain showed poor tolerance of NaCl and a narrower pH range for growth than R. lacus KACC 11678T and R. terrae KACC 12677T. However, strain 5N26T had a broader range of growth temperature than these two species. It showed white-coloured colonies while R. lacus KACC 11678T and R. terrae KACC 12677T had yellow and reddish pink colonies, respectively. In addition, the novel isolate showed negative reactions for catalase and urease, while its closest relatives exhibited positive reactions for these enzymes. The detailed morphological, physiological and biochemical characteristics of strain 5N26T and other species of the genus Roseomonas are shown in Table 1. Strain 5N26T had three major fatty acids, C16 : 0 (7.5 %), C18 : 1 2-OH (13.4 %) and summed feature 8 (C18 : 1v6c
Table 1. Differential phenotypic characteristics of strain 5N26T and type strains of closely related species Strains: 1, 5N26T (Roseomonas soli sp. nov.); 2, R. lacus KACC 11678T; 3, R. terrae KACC 12677T; 4, R. gilardii KACC 11652T; 5, R. cervicalis KACC 11686T; 6, R. ludipueritiae KACC 13843T. All data were obtained from this study. All strains are positive for aerobic growth and for alkaline phosphatase, esterase (C4), esterase lipase (C8), acid phosphatase and naphthol-AS-BI-phosphohydrolase activities, but negative for growth on MacConkey agar, hydrolysis of starch, CM-cellulose, Tween 80, chitin, hypoxanthine, xanthine and aesculin, valine arylamidase, cystine arylamidase, trypsin, lipase (C14), a-chymotrypsin, b-glucuronidase, a-galactosidase, b-galactosidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase, a-fucosidase, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase and tryptophan deaminase activities, H2S production, fermentation/oxidation of D-glucose, D-mannitol, inositol, D-sorbitol, L-rhamnose, sucrose, melibiose, amygdalin and glucose, and assimilation of D-mannose, N-acetylglucosamine, maltose, gluconate, caprate, adipate, malate, citrate and phenylacetate. +, Positive; (+), weakly positive; 2, negative. Characteristic Habitat Temperature range for growth (uC) Colony colour Catalase activity Hydrolysis of: Urea Casein Gelatin Assimilation of: D-Arabinose L-Arabinose
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1
2
3
4
5
6
Cabbage rhizosphere 15–40 White 2
Soil
Soil 4–30 Reddish pink +
Flood-plain sediment 4–30 Reddish pink +
Soil
10–30 Yellow +
Cabbage rhizosphere 4–35 Pinkish yellow +
4–30 Reddish pink +
2 2 2
+ 2 2
+ + +
+ + +
+ + +
+ + +
(+) 2
2 +
2 2
2 +
2 +
2 2
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D.-U. Kim and J.-O. Ka
Table 2. Cellular fatty acid contents of strain 5N26T and related species Strains: 1, 5N26T (Roseomonas soli sp. nov.); 2, R. lacus KACC 11678T; 3, R. terrae KACC 12677T; 4, R. gilardii KACC 11652T; 5, R. cervicalis KACC 11686T; 6, R. ludipueritiae KACC 13843T. All data were obtained from this study. Prior to fatty acid extraction, all strains were grown on R2A agar (Difco) at 30 uC for 2 days. Values are percentages of total fatty acids, and only fatty acids representing more than 1 % for at least one of the strains are shown. –, Not detected; TR, trace amounts (,1 %). Fatty acid C12 : 0 C14 : 0 C14 : 0 2-OH C16 : 0 C16 : 0 2-OH C16 : 0 3-OH C16 : 1v5c C18 : 0 C18 : 1 2-OH 11-Methyl C18 : 1v7c C19 : 0 cyclo v7c Summed feature 3* Summed feature 4* Summed feature 8*
1
2
–
– – – 4.6 –
TR
– 7.5 – 1.6 2.4 – 13.4 – 4.4 4.1 1.3 63.2
TR
3.3
3
4
–
–
TR
TR
– 9.4 1.7 1.3 2.6
TR
TR
7.6 4.8 2.2 4.3 – 70.3
12.3
– 25.0 TR
1.1 – 1.1 1.7
TR
TR
11.2 1.1 – 57.5
35.8 1.1 – 31.0
5
6
1.0 – 1.1 2.2 – 2.4 15.3 14.3 – TR 1.1 1.5 2.6 2.4 – – 3.1 2.3 – – 16.4 23.2 3.1 4.9 – – 55.4 43.6
*Summed features are groups of two or three fatty acids that cannot be separated using the MIDI system. Summed feature 3 comprises C16 : 1v7c and/or C16 : 1v6c; summed feature 4 comprises iso-C17 : 1 I and/or anteisoC17 : 1 B and summed feature 8 comprises C18 : 1v6c and/or C18 : 1v7c.
and/or C18 : 1v7c; 63.2 %), which are frequently found in species of the genus Roseomonas. The strain also had summed feature 4 (iso-C17 : 1 I and/or anteiso B) which was not found in other species of the genus Roseomonas. The
(a)
differential fatty acid characteristics of strain 5N26T and other species of the genus Roseomonas are shown in Table 2. The polar lipids present in 5N26T were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine (PE), phosphatidylcholine, phosphatidylmonomethylethanolamine and one unidentified aminolipid (Fig. 1). Previously, phosphatidylmonomethylethanolamine was detected in the polar lipid profiles of other species of the genus Roseomonas such as R. lacus CIP 109168T, R. mucosa CIP 108268T and R. cervicalis CIP 104027T (Sa´nchez-Porro et al., 2009). When the PE spot in the molybdenum blue image is compared with the corresponding spot in the ninhydrin image, the former is smaller than the latter. Another aminolipid spot (Fig. 1b, arrow) which stains only with ninhydrin and not with molybdenum blue might be present at the lower right part of the PE spot. But due to the high similarity in its chromatographic motility to that of PE it cannot be separated from PE. The presence of the second aminolipid has been reported in species of the genus Roseomonas (Sa´nchez-Porro et al., 2009). The polar lipid data showed that strain 5N26T belonged to the genus Roseomonas and additionally it appeared to have two other aminolipids including phosphatidylmonomethylethanolamine in its polar lipid pattern, making it novel among species of the genus Roseomonas. The DNA G+C content of strain 5N26T was 68.3 mol% and ubiquinone 10 (Q-10) was the predominant isoprenoid quinone. The sequence analyses of the 16S rRNA gene showed that strain 5N26T was closely related to R. lacus TH-G33T and R. terrae DS-48T. A previously reported 1376 bp 16S rRNA gene sequence of R. lacus TH-G33T had 41 nt differences from 5N26T. R. terrae DS-48T had 47 nt differences in 1414 bp of 16S rRNA gene sequence. The 16S rRNA gene sequence similarities of 5N26T were 97.0 % and 96.6 % with R. lacus TH-G33 T and R. terrae DS-48T, respectively. The neighbour-joining tree based on 16S rRNA gene sequences showed that strain 5N26T was grouped with the
(b) DPG PME
PME
PE
PE
PG AL PC
(c) DPG
PME PE
PG
AL PC
Fig. 1. Polar lipid profile of strain 5N26T after two-dimensional TLC, detected using molybdatophosphoric acid (a), ninhydrin (b) and molybdenum blue (c). PE, phosphatidylethanolamine; PME, phosphatidylmonomethylethanolamine; DPG, diphosphatidylglycerol; PG, phosphatidylglycerol; PC, phosphatidylcholine; AL, unknown aminolipid. Arrow, aminolipid spot which stains only with ninhydrin and not with molybdenum blue. 1026
International Journal of Systematic and Evolutionary Microbiology 64
Roseomonas soli sp. nov.
0.02
99
Roseomonas aestuarii JC17T (FM244739)
67
Roseomonas cervicalis ATCC 49957T (AY150047)
53
Roseomonas ludipueritiae 170/96T (AJ488504) Roseomonas aquatica TR53T (AM231587) Roseomonas rosea 173/96T (AJ488505)
77
Roseomonas pecuniae N75T (GU168019)
90
Roseomonas vinacea CPCC 100056T (EF368368) 99
59
Roseomonas aerilata 5420S-30T (EF661571)
T 72 Roseomonas gilardii subsp. rosea ATCC BAA-691 (AY220740) 100 Roseomonas gilardii subsp. gilardii ATCC 49956T (AY150045)
Roseomonas mucosa ATCC BAA-692T (AF538712) 52
Roseomonas riguiloci 03SU10-PT (HQ436503) Roseomonas stagni HS-69T (AB369258)
99 65
Roseomonas frigidaquae CW67T (EU290160) Roseomonas soli 5N26T (JN575264)
100
Roseomonas lacus TH-G33T (AJ786000) 89
Roseomonas terrae DS-48T (EF363716) Roseomonas fauriae ATCC 49958T (AY150046)
Fig. 2. Phylogenetic dendrogram reconstructed from a comparative analysis of 16S rRNA gene sequences showing the relationship between strain 5N26T and related species. Bootstrap values (expressed as percentages of 1000 replications) greater than 50 % are shown at branch points. Filled circles indicate that the corresponding branches were also recovered in the maximum-parsimony tree. Bar, 0.02 substitutions per nucleotide position.
members of the genus Roseomonas and formed a distinct phylogenetic line distinguishable from other species of the genus Roseomonas (Fig. 2). In conclusion, the main characteristics of strain 5N26T were in accordance with the description of the genus Roseomonas, but the new isolate could be distinguished from the other species of the genus Roseomonas by a combination of physiological and biochemical features and minor differences in their fatty acid profiles. Therefore, on the basis of the data from the polyphasic study presented here, strain 5N26T represents a novel species in the genus Roseomonas, for which the name Roseomonas soli sp. nov. is proposed. Description of Roseomonas soli sp. nov. Roseomonas soli (so9li. L. gen. n. soli of soil, the source of the type strain). Cells are Gram-reaction-negative, strictly aerobic, motile, non-spore-forming rods, 0.7–1.0 mm in diameter and 1.9– 2.4 mm in length after cultivation for 5 days at 30 uC on R2A agar. Colonies are 0.4–1.0 mm in diameter, smooth, circular, convex and white in colour after cultivation for 5 days at 30 uC on R2A agar. Grows on LB, NA, R2A agar, TSA and BCYE agar, but not on MacConkey agar. Catalase, urease and oxidase are negative. Growth occurs at 15–40 uC (optimal growth occurs at 30 uC) and pH 5.0–8.5 (optimal growth occurs at pH 6.5), but no growth occurs with 1 % NaCl. Does http://ijs.sgmjournals.org
not hydrolyse casein, gelatin, starch, tyrosine, Tween 80, chitin, CM-cellulose, hypoxanthine, urea, xanthine or aesculin. Positive (in API ZYM strips) for alkaline phosphatase, esterase (C4), esterase lipase (C8), acid phosphatase and naphthol-AS-BI-phosphohydrolase, and negative for leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, lipase (C14), a-chymotrypsin, b-glucuronidase, a-galactosidase, b-galactosidase, a-glucosidase, b-glucosidase, N-acetylb-glucosaminidase, a-mannosidase and a-fucosidase. Acetoin production is positive but negative for arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, citrate utilization, H2S production, tryptophan deaminase, indole production and gelatinase. Fermentation/oxidation of D-glucose, D-mannitol, inositol, D-sorbitol, L-rhamnose, sucrose, melibiose, amygdalin and D-arabinose are negative (in API 20E strips). Acidification of glucose is negative and assimilation of D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetylglucosamine, maltose, gluconate, caprate, adipate, malate, citrate and phenylacetate are negative (in API 20NE strips). Ubiquinone 10 (Q-10) is the predominant quinone. The major fatty acids are C16 : 0, C18 : 1 2-OH and summed feature 8 (C18 : 1v6c and/or C18 : 1 v7c). The polar lipids are diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, phosphatidylmonomethylethanolamine and one unidentified aminolipid. The type strain is 5N26T (5KACC 16376T5NBRC 109097T), isolated from rhizosphere soil cultivated with Chinese cabbage (Brassica campestris L.) in Namyangju, 1027
D.-U. Kim and J.-O. Ka
Korea. The DNA G+C content of the type strain is 68.3 mol%.
Acknowledgements This work was supported by a grant from the Regional SubGenBank Support Program of Rural Development Administration, Republic of Korea.
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