Antonie van Leeuwenhoek (2014) 105:191–197 DOI 10.1007/s10482-013-0065-4
ORIGINAL PAPER
Roseomonas sediminicola sp. nov., isolated from fresh water Dan He • Jin-Kwang Kim • Xiao-Ye Jiang • Hye-Yoon Park Changkai Sun • Hong-San Yu • Min-Ho Yoon • Sun-Chang Kim • Feng Xie Jin • Wan-Taek Im
•
Received: 31 May 2013 / Accepted: 24 October 2013 / Published online: 28 November 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract A Gram-stain negative, strictly aerobic, non-motile, non-spore-forming, and rod-shaped bacterial strain designated FW-3T was isolated from fresh water and its taxonomic position was investigated by using a polyphasic approach. Strain FW-3T was found to grow at 10–37 °C and at pH 7.0 in the absence of NaCl on nutrient agar. On the basis of 16S rRNA gene sequence similarity, strain FW-3T was shown to belong to the family Acetobacteraceae and to be related to Roseomonas lacus TH-G33T (97.2 % sequence similarity) and Roseomonas terrae DS-48T (96.4 %). The G?C content of the genomic DNA was determined to be 68.0 %. The major menaquinone was determined to be Q-10 and the major fatty acids were identified as
summed feature 7 (comprising C18:1 x9c/x12t/x7c as defined by the MIDI system; 55.4 %), and C18:1 2OH (29.8 %). DNA and chemotaxonomic data supported the affiliation of strain FW-3T to the genus Roseomonas. Strain FW-3T could be differentiated genotypically and phenotypically from the recognized species of the genus Roseomonas. The novel isolate therefore represents a novel species, for which the name Roseomonas sediminicola sp. nov. is proposed, with the type strain FW-3T (=KACC 16616T = JCM 18210T).
D. He J.-K. Kim S.-C. Kim W.-T. Im (&) KI for the Biocentury, Korea Advanced Institute of Science and Technology, 373-1, Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea e-mail: [email protected]
H.-Y. Park Microorganism Resources Division, National Institute of Biological Resources, Incheon 404-708, Republic of Korea
D. He X.-Y. Jiang H.-S. Yu F. X. Jin College of Biotechnology, Dalian Polytechnic University, Qinggong-yuan No. 1, Ganjingzi-qu, Dalian 116034, People’s Republic of China e-mail: [email protected]
Keywords 16S rRNA gene Polyphasic taxonomy Roseomonas sediminicola
C. Sun Institute for Brain Disorders, Dalian Medical University, Dalian 116044, People’s Republic of China
J.-K. Kim M.-H. Yoon Department of Bio-Environmental Chemistry, College of Agriculture and Life Sciences, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejeon 305-764, Republic of Korea
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Introduction During the course of a study on the culturable aerobic bacterial community from the fresh water of a duck lake, KAIST, Daejeon, South Korea, several novel bacterial strains were screened. One of these isolates, strain FW-3T, was indicated to be a member of the genus Roseomonas and was subjected to a taxonomic investigation. The genus Roseomonas was created as a novel member of the family Acetobacteraceae by Rihs et al. (1993) with the description of three species, Roseomonas gilardii, Roseomonas cervicalis and Roseomonas fauriae. They are pink-coloured, Gramnegative and coccoid-shaped. At the time of writing, the genus consisted of 18 validly named species, with R. gilardii as the type species (Euze´by 1997, http:// www.bacterio.net/qr/roseomonas.html), including recently recognized species such as R. aestuarii (Venkata Ramana et al. 2010), R. pecuniae (Lopes et al. 2011), R. riguiloci (Baik et al. 2012), R. aerophila (Kim et al. 2013), and R. musae (Nutaratat et al. 2013). These were isolated from various environments such as clinical specimens, water, marine invertebrates, soil, air and the plant phyllosphere. In the present study, we conducted phylogenetic (16S rRNA gene), phenotypic, genotypic, and chemotaxonomic analyses to determine the taxonomic position of this strain. On the basis of the results obtained in this study, we propose that strain FW-3T should be placed in the genus Roseomonas as the type strain of a novel species, Roseomonas sediminicola sp. nov.
Materials and methods Isolation of bacterial strain and culture conditions Strain FW-3T was originally isolated from freshwater (pH 7.2, 24 °C) of a duck lake in KAIST, Daejeon, South Korea. This sediment sample was thoroughly suspended in 50 mM phosphate buffer (pH 7.0) and spread on nutrient agar (NA, Difco, USA) plates. The plates were incubated at 30 °C for 2 weeks. One isolate, FW-3T, was cultured routinely on R2A agar (Difco, USA) or NA at 25 °C and preserved as a suspension in nutrient broth with 20 % (w/v) glycerol
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at -70 °C. Strain FW-3T was deposited in the Korean Agricultural Culture Collection (=KACC 16616T) and the Japan Collection of Microorganisms (=JCM 18210T). The reference strains Roseomonas lacus KACC 11678T and Roseomonas terrae KACC 12677T were obtained from KACC and cultured under comparable conditions for comparative testing. Phenotypic and biochemical characteristics The Gram reaction was determined using the nonstaining method using 3 % KOH, as described by Buck (1982). Cell morphology and motility was observed under a Nikon light microscope at 1,0009, with cells grown on R2A agar for 2 days at 30 °C. Catalase and oxidase tests were performed as described by Cappuccino and Sherman (2002). Anaerobic growth was determined in serum bottles containing R2A broth supplemented with thioglycolate (1 g/l), in which the upper air layer had been replaced with nitrogen. In addition, biochemical tests were carried out using API 20NE, API ID 32GN and API ZYM test kits according to the instructions of the manufacturer (bioMe´rieux, France). Tests for degradation of DNA (using DNase agar from Scharlau, with DNase activity detected by flooding plates with 1 M HCl), casein, and starch (Atlas 1993) were performed and evaluated after 7 days. Growth at different temperatures (4, 10, 18, 30, 37, 42 and 45 °C) and various pH values (pH 4.5–10.0 at intervals of 0.5 pH units) was assessed after 7 days of incubation in R2A broth. Three different buffers (final concentration, 50 mM) were used to adjust the pH of R2A broth. Acetate buffer was used for pH 5.0–5.5, phosphate buffer was used for pH 6.0–8.0 and Tris buffer was used for pH 8.5–10.0. Salt tolerance was tested on R2A medium supplemented with 1–10 % (w/ v at intervals of 1 % unit) NaCl and growth assessed after 7 days of incubation. Growth on nutrient agar, trypticase soy agar (TSA, Difco) and MacConkey agar (Difco) was also evaluated at 30 °C. Susceptibility to antibiotics was tested on R2A plates using antibiotic discs containing the following: ampicillin, 10 lg; cephalothin, 30 lg; gentamycin, 10 lg; kanamycin, 30 lg; streptomycin, 10 lg; erythromycin, 15 lg; chloramphenicol, 30 lg; tetracycline, 30 lg; lincomycin, 15 lg; neomycin, 30 lg; penicillin, 10 U; vancomycin, 30 lg; cycloheximide, 10 lg; novobiocin, 10 lg; oleandomycin, 10 lg.
Antonie van Leeuwenhoek (2014) 105:191–197
PCR amplification, 16S rRNA gene sequencing and phylogenetic analysis The genomic DNA of strain FW-3T was extracted using a commercial genomic DNA extraction kit (Solgent, Korea). The 16S rRNA gene was amplified from the chromosomal DNA using the universal bacterial primer pair 9F and 1512R and the purified PCR products were sequenced by Solgent Co. Ltd (Daejeon, Korea) (Im et al. 2010). Full sequences of the 16S rRNA gene were compiled using SeqMan software (DNASTAR, USA). The 16S rRNA gene sequences of related taxa were obtained from GenBank and the EzTaxon-e server (Kim et al. 2012). Multiple alignments were performed using the Clustal_X program (Thompson et al. 1997) and gaps were edited in the BioEdit program (Hall 1999). Evolutionary distances were calculated using the Kimura twoparameter model (Kimura 1983). The phylogenetic trees were constructed by using the neighbour-joining (Saitou and Nei 1987) and the maximum-parsimony (Fitch 1971) methods with the MEGA4 Program (Kumar et al. 2008) with bootstrap values based on 1,000 replications (Felsenstein 1985).
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extracted and purified as described by Moore and Dowhan (1995) and enzymatically degraded into nucleosides. The G?C content was then determined as described by Mesbah et al. (1989), using reversephase HPLC. DNA–DNA hybridization DNA–DNA hybridization experiments were performed between strain FW-3T and the two reference strains R. lacus KACC 11678T and R. terrae KACC 12677T using the method described by Ezaki et al. (1989) using photobiotin-labeled DNA probes and micro-dilution wells. Hybridizations were performed at 52 °C with five replications for each sample. The highest and lowest values obtained for each sample were excluded and the means of the remaining three values were converted to percentage DNA–DNA relatedness values.
Results and discussion Morphological and phenotypic characteristics
Isoprenoid quinones were extracted with chloroform/ methanol (2:1 v/v), evaporated under a vacuum and reextracted in n-hexane–water (1:1 v/v). The crude quinone in n-hexane solution was purified using SepPak Vac Cartridges Silica (Waters) and subsequently analyzed by HPLC, as described by Hiraishi et al. (1996). For cellular fatty acid analysis, strain FW-3T and the two reference type strains (R. lacus KACC 11678T and R. terrae KACC 12677T) were grown at 30 °C for 48 h on R2A agar until they reached the exponential phase. The cellular fatty acids were saponified, methylated and extracted according to the protocol of the Sherlock Microbial Identification System (MIDI). The fatty acid methyl esters were then analyzed by gas chromatography (model 6,890; Hewlett Packard) using the Microbial Identification software package (Sasser 1990).
The cells of strain FW-3T were observed to be Gramstain negative, strictly aerobic, non-spore-forming, non-motile, rod shaped, oxidase-positive and catalasepositive. The colonies grown on R2A agar plates for 2 days were smooth, circular, transparent, pale red in colour, convex, and 2–3 mm in diameter. On R2A agar, FW-3T was able to grow at 10–37 °C, but not at 42 °C. The isolate was able to grow on nutrient agar and TSA, but not on MacConkey agar. Strain FW-3T was found to be susceptible to (lg per disc, unless otherwise indicated) cephalothin (30), kanamycin (30), streptomycin (10), erythromycin (15), chloroamphenicol (30), tetracycline (30), neomycin (30), and oleandomycin (10), but not to ampicillin (10), gentamycin (10), lincomycin (15), penicillin (10U), vancomycin (30), cycloheximide (10), and novobiocin (10). The phenotypic characteristics that differentiate the strain FW-3T from the most closely related Roseomonas species are listed in Table 1.
Determination of DNA G?C content
Phylogenetic analysis
For measurement of the G?C content of chromosomal DNA, the genomic DNA of strain FW-3T was
The 16S rRNA gene sequence of the strain FW-3T determined in this study is a continuous stretch of
Isoprenoid quinone, cellular fatty acids analysis
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Table 1 Differentiating characteristics of FW-3T and the type strains of related Roseomonas species Characteristics Isolation source
1
2
3
Freshwater
Freshwater
Soil
sediment
sediment
Cell morphology
Rod
Coccoid rods
Coccobacilli
Motility
–
–
–
Colony color
Pale red
Colorless
Pale yellow
pale pink
to pale pink 10–30
Temperature range (°C)
10–37
10–37
pH range
5.5–10
5–10
5–10
NaCl range (%)
1
1
1–2
Arginine dihydrolase
–
?
–
Urease
?
?
–
Decomposition of:
1,409 bp (base position 28-1491 with respect to the Escherichia coli numbering system), which has been deposited in the GenBank database (accession number JQ349047). A sequence similarity calculation from using the EzTaxon-e server (http://eztaxon-e. ezbiocloud.net; Kim et al. 2012) indicated that the closest relatives of strain FW-3T were R. lacus THG33T (97.2 % sequence similarity) and R. terrae DS48T (96.4 %). This relationship between strain FW-3T and members of the genus Roseomonas was also evident in the phylogenetic tree, which used over 1,350 nt (Fig. 1). Strain FW-3T, R. lacus TH-G33T and R. terrae DS-48T formed a monophyletic group with a bootstrap high value (100 %), which was supported by both tree making methods used in this study.
DNA
–
?
–
Skim milk
–
?
–
Quinone and cellular fatty acids composition
Nitrate reduction
?
?
–
Gluconate
–
–
?
The major respiratory quinone of strain FW-3T was determined to be ubiquinone 10 (Q-10), in line with all other members of the family Acetobacteraceae. The fatty acid profile of strain FW-3T (i.e. those fatty acids constituting[1.0 % of the total) included unsaturated fatty acids in summed feature 7 as defined by the MIDI system (comprising C18:1 x9c/x12t/x7c; 55.4 %) and C17:1 x7c (1.1 %), hydroxyl fatty acids C18:1 2OH (29.8 %), straight-chain fatty acids C16:0 (8.9 %) and C12:0 (1.5 %), cyclo fatty acids C19:0 cyclo x8c (1.2 %) and summed feature 3 (comprising C14:0 3OH and/or iso-C16:1; 1.3 %) (Table 2). This fatty acid profile was similar to those of Roseomonas species. However, there were differences between strain FW-3T and its phylogenetically closest relatives, R. lacus KACC 11678T and R. terrae KACC 12677T, in terms of the contents of the predominant fatty acids, i.e. summed feature 7 and C18:1 2OH. Furthermore, the absence of two hydroxyl fatty acids (C16:0 2OH and C16:0 3OH) clearly differentiated strain FW-3T from the two closest neighbours.
Carbon utilization of
Adipate
–
?
?
Malate
?
–
?
–
?
–
Propionate
L-Arabinose
–
?
?
Valerate
–
?
?
Citrate
–
?
–
L-Proline
?
?
–
D-Ribose
?
–
–
Malonate
–
?
?
Acetate
?
–
–
5-Ketogluconate
?
?
–
Glycogen
–
?
–
API ZYM test results Trypsin
?
–
–
Acid phosphatase
–
?
?
G?C content (mol%)
68.0
71.9
69.3
T
Strains: 1, Roseomonas sediminicola FW-3 ; 2, Roseomonas lacus KACC 11678T; 3, Roseomonas terrae KACC 12677T. All data from this study, except the DNA G?C contents of the reference strains (taken from Jiang et al. 2006; Yoon et al. 2007). All strains are Gram-reaction-negative, positive for catalase, and oxidase activities but negative for hydrolysis of xylan, starch, Tween 80, tyrosine and casein. In API 20 NE and API ID 32 GN kits all strains are positive for 2-ketogluconate, 3-hydroxy-butyrate, suberate, and L-alanine activities. All strains are negative for reduction of nitrate to nitrogen, indole production, acid production from glucose, and assimilation of the following substrates: D-mannitol, D-glucose, salicin, Dmelibiose, L-fucose, D-sorbitol, caprate, L-histidine, 4-hydroxy-benzoate, Lrhamnose, N-acetyl-D-glucosamine, inositol, D-sucrose, D-maltose, itaconate, lactate, 3-hydroxy-benzoate, and L-serine. In API ZYM kits, all the strains are positive for alkaline phosphatase, esterase (C4), and esterase lipase (C8) activities but negative for lipase (C14), leucine arylamidase, valine arylamidase, crystine arylamidase, a-chymotrypsin, naphtol-AS-BIphosphohydrolase, a-galactosidase, b-galactosidase, b-glucuronidase, aglucosidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase, and a-fucosidase. ? Positive; – negative
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DNA G?C content and DNA–DNA hybridization The DNA G?C content of strain FW-3T was determined to be 68.0 mol%, a value comparable to those of R. lacus TH-G33T (71.9 %; Jiang et al. 2006) and R. terrae DS-48T (69.3 %; Yoon et al. 2007) and in the middle of range when compared to the other species in the genus Roseomonas (65.8–72.1 mol %). DNA– DNA relatedness values between strain FW-3T and R.
Antonie van Leeuwenhoek (2014) 105:191–197
195 Roseomonas lacus TH-G33T (AJ786000)
95 100
Roseomonas terrae DS-48T (EF363716)
Roseomonas sediminicola sp. nov. FW-3T (JQ349047) Roseomonas aquatica TR53T (AM231587) Roseomonas ludipueritiae 170/96T (AJ488504) Roseomonas aestuarii JC17T (FM244739)
100
Roseomonas cervicalis ATCC 49957T (AY150047) Roseomonas frigidaquae CW67T (EU290160) Roseomonas stagni HS-69T (AB369258)
100
96 100
Roseomonas gilardii subsp. gilardii ATCC 49956T (AY150045) Roseomonas gilardii subsp. rosea MDA5605T (AY220740) Roseomonas mucosa MDA5527T (AF538712) Roseomonas rosea 173/96T (AJ488505) Roseomonas pecuniae N75T (GU168019)
80
Roseomonas aerilata 5420S-30T (EF661571) 98
Roseomonas vinacea CPCC 100056T (EF368368) Craurococcus roseus NS130T (D85828) Paracraurococcus ruber NS89T (D85827)
84
Belnapia moabensis CP2CT (AJ871428) Roseococcus suduntuyensis SHETT (EU012448) Rubritepida flocculans DSM 14296T (AF465832)
100
Rhodovarius lipocyclicus CCUG 44693T (AJ633644) Acidisphaera rubrifaciens HS-AP3T (D86512)
98 69
Rhodovastum atsumiense G211T (AB381935) Acetobacter aceti NCIMB 8621T (X74066)
89
Gluconobacter cerinus IFO 3267T (X80775)
100 100
Gluconobacter oxydans NBRC 14819T (AB178433) Azospirillum brasilense ATCC 49958T (AY150046)
0.02
Fig. 1 Phylogenetic tree constructed from a comparative analysis of 16S rRNA gene sequences showing the relationships of Roseomonas sediminicola FW-3T with other related species. This tree was made using the neighbour-joining method (Saitou and Nei 1987) with a Kimura (1983) two-parameter distance
matrix and pairwise deletion. Dots indicate branches that were also recovered by using the maximum-parsimony algorithm. Bootstrap values (expressed as percentages of 1,000 replications) greater than 70 % are shown at the branch points. Bar 0.01 substitutions per 1 nucleotide position
lacus KACC 11678T and R. terrae KACC 12677T were 5 ± 1.2 and 4 ± 1.7 %, respectively. According to Wayne et al. (1987), DNA–DNA relatedness values lower than 70 % are considered to be the threshold value for the delineation of genospecies, so the result obtained is low enough to assign strain FW-3T as a novel species of the genus Roseomonas.
phylogenetic analysis warrant the assignment of strain FW-3T to the genus Roseomonas as the type strain of a novel species, for which the name Roseomonas sediminicola sp. nov. is proposed.
Taxonomic conclusions The characteristics of strain FW-3T are consistent with the description of the genus Roseomonas with regard to morphological, biochemical and chemotaxonomic properties. However, the phylogenetic distance between strain FW-3T and recognized Roseomonas species, the unique phenotypic characteristics (Table 1) and the low level of DNA–DNA relatedness values with the nearest neighbours identified by
Description of Roseomonas sediminicola sp. nov. Roseomonas sediminicola (se.di.mi.ni.co0 la. L. n. sedimen, -inis sediment; L. suff. -cola inhabitant, dweller; N.L. n. sediminicola sediment-dweller, referring to the source of the type strain). Cells are Gram-stain negative, strictly aerobic, nonmotile and non-spore-forming rods (0.3–0.5 lm in diameter and 1.5–2.2 lm in length) after culture on R2A agar for 2 days. Colonies are smooth, transparent, convex, circular with regular margins, pale red in colour, and 2–3 mm in diameter after 2 days on R2A agar. Growth also occurs on nutrient agar and TSA,
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Antonie van Leeuwenhoek (2014) 105:191–197
Table 2 Cellular fatty acid profiles of strain FW-3T and phylogenetically related species of the genus Roseomonas Fatty acid
1
2
3
C12:0
1.5
–
–
C16:0
8.9
8.0
11.1
C16:1 x5c
0.9
2.2
2.6
C17:1 x7c
1.1
–
–
Saturated
Acknowledgments This work was supported by the Intelligent Synthetic Biology Center of Global Frontier Project funded by the Ministry of Education, Science and Technology (2011-0031967) and by the project on survey and excavation of Korean indigenous species of the National Institute of Biological Resources (NIBR) under the Ministry of Environment, Korea and by the China Major Projects No.2012ZX09503001-003.
Unsaturated
Hydroxy fatty acids C16:0 2OH
–
1.7
4.0
C16:0 3OH
–
1.5
4.3
C18:1 2OH
29.8
8.7
20.7
1.2
5.0
11.4
Cyclo fatty acid C19:0 cyclo x8c Summed feature 3; C14:0 3OH/iso-C16:1
1.3
–
–
4; C16:1 x7c/iso-C15:0 2OH 7; C18:1 x9c/x12t/x7c
– 55.4
4.6 68.2
– 45.9
Summed feature represent groups of two or three fatty acids that could not be separated by GLC with the MIDI system. Summed feature consist of: 3, C16:1 x7c/C16:1 x6c; 8, C18:1 x7c/x6c; 4, C16:1 x7c/iso-C15:0 2OH; 7, C18:1 x9c/x12t/x7c. Strains: 1, Roseomonas sediminicola FW-3T; 2, Roseomonas lacus KACC 11678T; 3, Roseomonas terrae KACC 12677T. All data from this study. All strains were cultured on nutrient agar for 2 days at 30 °C. Fatty acids amounting to \0.5 % of the total fatty acids in all strains are not listed. -, not detected. tr, trace amount below 0.5 %
but not on MacConkey. Grows on R2A at 10–37 °C and at pH 5.5–10, but not at 42 °C. Optimum growth occurs at 25–37 °C and at pH 7.0. Growth occurs well without NaCl supplement and in the presence of 1.0 % (w/v) NaCl. Catalase and oxidase are positive. Does not hydrolyze casein, DNA, xylan, skim milk, tyrosine, tween 80, and starch. Carbon assimilation tests as a sole carbon sources (API ID 32 GN, API 20 NE) and the enzyme activities (API ZYM) are listed in Table 1. Q-10 is the predominant respiratory quinone, and summed feature 7 (comprising C18:1 x9c/x12t/x7c) and C18:1 2OH are the major cellular fatty acids. The G?C content of the genomic DNA of the type strain is 68.0 mol %. The type strain, FW-3T (=KACC 16616T = JCM 18210T) was isolated from the fresh water of duck lake, KAIST, Daejeon, South Korea. The GenBank accession number of the 16S rRNA gene is JQ349047.
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ORIGINAL PAPER
Roseomonas sediminicola sp. nov., isolated from fresh water Dan He • Jin-Kwang Kim • Xiao-Ye Jiang • Hye-Yoon Park Changkai Sun • Hong-San Yu • Min-Ho Yoon • Sun-Chang Kim • Feng Xie Jin • Wan-Taek Im
•
Received: 31 May 2013 / Accepted: 24 October 2013 / Published online: 28 November 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract A Gram-stain negative, strictly aerobic, non-motile, non-spore-forming, and rod-shaped bacterial strain designated FW-3T was isolated from fresh water and its taxonomic position was investigated by using a polyphasic approach. Strain FW-3T was found to grow at 10–37 °C and at pH 7.0 in the absence of NaCl on nutrient agar. On the basis of 16S rRNA gene sequence similarity, strain FW-3T was shown to belong to the family Acetobacteraceae and to be related to Roseomonas lacus TH-G33T (97.2 % sequence similarity) and Roseomonas terrae DS-48T (96.4 %). The G?C content of the genomic DNA was determined to be 68.0 %. The major menaquinone was determined to be Q-10 and the major fatty acids were identified as
summed feature 7 (comprising C18:1 x9c/x12t/x7c as defined by the MIDI system; 55.4 %), and C18:1 2OH (29.8 %). DNA and chemotaxonomic data supported the affiliation of strain FW-3T to the genus Roseomonas. Strain FW-3T could be differentiated genotypically and phenotypically from the recognized species of the genus Roseomonas. The novel isolate therefore represents a novel species, for which the name Roseomonas sediminicola sp. nov. is proposed, with the type strain FW-3T (=KACC 16616T = JCM 18210T).
D. He J.-K. Kim S.-C. Kim W.-T. Im (&) KI for the Biocentury, Korea Advanced Institute of Science and Technology, 373-1, Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea e-mail: [email protected]
H.-Y. Park Microorganism Resources Division, National Institute of Biological Resources, Incheon 404-708, Republic of Korea
D. He X.-Y. Jiang H.-S. Yu F. X. Jin College of Biotechnology, Dalian Polytechnic University, Qinggong-yuan No. 1, Ganjingzi-qu, Dalian 116034, People’s Republic of China e-mail: [email protected]
Keywords 16S rRNA gene Polyphasic taxonomy Roseomonas sediminicola
C. Sun Institute for Brain Disorders, Dalian Medical University, Dalian 116044, People’s Republic of China
J.-K. Kim M.-H. Yoon Department of Bio-Environmental Chemistry, College of Agriculture and Life Sciences, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejeon 305-764, Republic of Korea
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Introduction During the course of a study on the culturable aerobic bacterial community from the fresh water of a duck lake, KAIST, Daejeon, South Korea, several novel bacterial strains were screened. One of these isolates, strain FW-3T, was indicated to be a member of the genus Roseomonas and was subjected to a taxonomic investigation. The genus Roseomonas was created as a novel member of the family Acetobacteraceae by Rihs et al. (1993) with the description of three species, Roseomonas gilardii, Roseomonas cervicalis and Roseomonas fauriae. They are pink-coloured, Gramnegative and coccoid-shaped. At the time of writing, the genus consisted of 18 validly named species, with R. gilardii as the type species (Euze´by 1997, http:// www.bacterio.net/qr/roseomonas.html), including recently recognized species such as R. aestuarii (Venkata Ramana et al. 2010), R. pecuniae (Lopes et al. 2011), R. riguiloci (Baik et al. 2012), R. aerophila (Kim et al. 2013), and R. musae (Nutaratat et al. 2013). These were isolated from various environments such as clinical specimens, water, marine invertebrates, soil, air and the plant phyllosphere. In the present study, we conducted phylogenetic (16S rRNA gene), phenotypic, genotypic, and chemotaxonomic analyses to determine the taxonomic position of this strain. On the basis of the results obtained in this study, we propose that strain FW-3T should be placed in the genus Roseomonas as the type strain of a novel species, Roseomonas sediminicola sp. nov.
Materials and methods Isolation of bacterial strain and culture conditions Strain FW-3T was originally isolated from freshwater (pH 7.2, 24 °C) of a duck lake in KAIST, Daejeon, South Korea. This sediment sample was thoroughly suspended in 50 mM phosphate buffer (pH 7.0) and spread on nutrient agar (NA, Difco, USA) plates. The plates were incubated at 30 °C for 2 weeks. One isolate, FW-3T, was cultured routinely on R2A agar (Difco, USA) or NA at 25 °C and preserved as a suspension in nutrient broth with 20 % (w/v) glycerol
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at -70 °C. Strain FW-3T was deposited in the Korean Agricultural Culture Collection (=KACC 16616T) and the Japan Collection of Microorganisms (=JCM 18210T). The reference strains Roseomonas lacus KACC 11678T and Roseomonas terrae KACC 12677T were obtained from KACC and cultured under comparable conditions for comparative testing. Phenotypic and biochemical characteristics The Gram reaction was determined using the nonstaining method using 3 % KOH, as described by Buck (1982). Cell morphology and motility was observed under a Nikon light microscope at 1,0009, with cells grown on R2A agar for 2 days at 30 °C. Catalase and oxidase tests were performed as described by Cappuccino and Sherman (2002). Anaerobic growth was determined in serum bottles containing R2A broth supplemented with thioglycolate (1 g/l), in which the upper air layer had been replaced with nitrogen. In addition, biochemical tests were carried out using API 20NE, API ID 32GN and API ZYM test kits according to the instructions of the manufacturer (bioMe´rieux, France). Tests for degradation of DNA (using DNase agar from Scharlau, with DNase activity detected by flooding plates with 1 M HCl), casein, and starch (Atlas 1993) were performed and evaluated after 7 days. Growth at different temperatures (4, 10, 18, 30, 37, 42 and 45 °C) and various pH values (pH 4.5–10.0 at intervals of 0.5 pH units) was assessed after 7 days of incubation in R2A broth. Three different buffers (final concentration, 50 mM) were used to adjust the pH of R2A broth. Acetate buffer was used for pH 5.0–5.5, phosphate buffer was used for pH 6.0–8.0 and Tris buffer was used for pH 8.5–10.0. Salt tolerance was tested on R2A medium supplemented with 1–10 % (w/ v at intervals of 1 % unit) NaCl and growth assessed after 7 days of incubation. Growth on nutrient agar, trypticase soy agar (TSA, Difco) and MacConkey agar (Difco) was also evaluated at 30 °C. Susceptibility to antibiotics was tested on R2A plates using antibiotic discs containing the following: ampicillin, 10 lg; cephalothin, 30 lg; gentamycin, 10 lg; kanamycin, 30 lg; streptomycin, 10 lg; erythromycin, 15 lg; chloramphenicol, 30 lg; tetracycline, 30 lg; lincomycin, 15 lg; neomycin, 30 lg; penicillin, 10 U; vancomycin, 30 lg; cycloheximide, 10 lg; novobiocin, 10 lg; oleandomycin, 10 lg.
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PCR amplification, 16S rRNA gene sequencing and phylogenetic analysis The genomic DNA of strain FW-3T was extracted using a commercial genomic DNA extraction kit (Solgent, Korea). The 16S rRNA gene was amplified from the chromosomal DNA using the universal bacterial primer pair 9F and 1512R and the purified PCR products were sequenced by Solgent Co. Ltd (Daejeon, Korea) (Im et al. 2010). Full sequences of the 16S rRNA gene were compiled using SeqMan software (DNASTAR, USA). The 16S rRNA gene sequences of related taxa were obtained from GenBank and the EzTaxon-e server (Kim et al. 2012). Multiple alignments were performed using the Clustal_X program (Thompson et al. 1997) and gaps were edited in the BioEdit program (Hall 1999). Evolutionary distances were calculated using the Kimura twoparameter model (Kimura 1983). The phylogenetic trees were constructed by using the neighbour-joining (Saitou and Nei 1987) and the maximum-parsimony (Fitch 1971) methods with the MEGA4 Program (Kumar et al. 2008) with bootstrap values based on 1,000 replications (Felsenstein 1985).
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extracted and purified as described by Moore and Dowhan (1995) and enzymatically degraded into nucleosides. The G?C content was then determined as described by Mesbah et al. (1989), using reversephase HPLC. DNA–DNA hybridization DNA–DNA hybridization experiments were performed between strain FW-3T and the two reference strains R. lacus KACC 11678T and R. terrae KACC 12677T using the method described by Ezaki et al. (1989) using photobiotin-labeled DNA probes and micro-dilution wells. Hybridizations were performed at 52 °C with five replications for each sample. The highest and lowest values obtained for each sample were excluded and the means of the remaining three values were converted to percentage DNA–DNA relatedness values.
Results and discussion Morphological and phenotypic characteristics
Isoprenoid quinones were extracted with chloroform/ methanol (2:1 v/v), evaporated under a vacuum and reextracted in n-hexane–water (1:1 v/v). The crude quinone in n-hexane solution was purified using SepPak Vac Cartridges Silica (Waters) and subsequently analyzed by HPLC, as described by Hiraishi et al. (1996). For cellular fatty acid analysis, strain FW-3T and the two reference type strains (R. lacus KACC 11678T and R. terrae KACC 12677T) were grown at 30 °C for 48 h on R2A agar until they reached the exponential phase. The cellular fatty acids were saponified, methylated and extracted according to the protocol of the Sherlock Microbial Identification System (MIDI). The fatty acid methyl esters were then analyzed by gas chromatography (model 6,890; Hewlett Packard) using the Microbial Identification software package (Sasser 1990).
The cells of strain FW-3T were observed to be Gramstain negative, strictly aerobic, non-spore-forming, non-motile, rod shaped, oxidase-positive and catalasepositive. The colonies grown on R2A agar plates for 2 days were smooth, circular, transparent, pale red in colour, convex, and 2–3 mm in diameter. On R2A agar, FW-3T was able to grow at 10–37 °C, but not at 42 °C. The isolate was able to grow on nutrient agar and TSA, but not on MacConkey agar. Strain FW-3T was found to be susceptible to (lg per disc, unless otherwise indicated) cephalothin (30), kanamycin (30), streptomycin (10), erythromycin (15), chloroamphenicol (30), tetracycline (30), neomycin (30), and oleandomycin (10), but not to ampicillin (10), gentamycin (10), lincomycin (15), penicillin (10U), vancomycin (30), cycloheximide (10), and novobiocin (10). The phenotypic characteristics that differentiate the strain FW-3T from the most closely related Roseomonas species are listed in Table 1.
Determination of DNA G?C content
Phylogenetic analysis
For measurement of the G?C content of chromosomal DNA, the genomic DNA of strain FW-3T was
The 16S rRNA gene sequence of the strain FW-3T determined in this study is a continuous stretch of
Isoprenoid quinone, cellular fatty acids analysis
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Table 1 Differentiating characteristics of FW-3T and the type strains of related Roseomonas species Characteristics Isolation source
1
2
3
Freshwater
Freshwater
Soil
sediment
sediment
Cell morphology
Rod
Coccoid rods
Coccobacilli
Motility
–
–
–
Colony color
Pale red
Colorless
Pale yellow
pale pink
to pale pink 10–30
Temperature range (°C)
10–37
10–37
pH range
5.5–10
5–10
5–10
NaCl range (%)
1
1
1–2
Arginine dihydrolase
–
?
–
Urease
?
?
–
Decomposition of:
1,409 bp (base position 28-1491 with respect to the Escherichia coli numbering system), which has been deposited in the GenBank database (accession number JQ349047). A sequence similarity calculation from using the EzTaxon-e server (http://eztaxon-e. ezbiocloud.net; Kim et al. 2012) indicated that the closest relatives of strain FW-3T were R. lacus THG33T (97.2 % sequence similarity) and R. terrae DS48T (96.4 %). This relationship between strain FW-3T and members of the genus Roseomonas was also evident in the phylogenetic tree, which used over 1,350 nt (Fig. 1). Strain FW-3T, R. lacus TH-G33T and R. terrae DS-48T formed a monophyletic group with a bootstrap high value (100 %), which was supported by both tree making methods used in this study.
DNA
–
?
–
Skim milk
–
?
–
Quinone and cellular fatty acids composition
Nitrate reduction
?
?
–
Gluconate
–
–
?
The major respiratory quinone of strain FW-3T was determined to be ubiquinone 10 (Q-10), in line with all other members of the family Acetobacteraceae. The fatty acid profile of strain FW-3T (i.e. those fatty acids constituting[1.0 % of the total) included unsaturated fatty acids in summed feature 7 as defined by the MIDI system (comprising C18:1 x9c/x12t/x7c; 55.4 %) and C17:1 x7c (1.1 %), hydroxyl fatty acids C18:1 2OH (29.8 %), straight-chain fatty acids C16:0 (8.9 %) and C12:0 (1.5 %), cyclo fatty acids C19:0 cyclo x8c (1.2 %) and summed feature 3 (comprising C14:0 3OH and/or iso-C16:1; 1.3 %) (Table 2). This fatty acid profile was similar to those of Roseomonas species. However, there were differences between strain FW-3T and its phylogenetically closest relatives, R. lacus KACC 11678T and R. terrae KACC 12677T, in terms of the contents of the predominant fatty acids, i.e. summed feature 7 and C18:1 2OH. Furthermore, the absence of two hydroxyl fatty acids (C16:0 2OH and C16:0 3OH) clearly differentiated strain FW-3T from the two closest neighbours.
Carbon utilization of
Adipate
–
?
?
Malate
?
–
?
–
?
–
Propionate
L-Arabinose
–
?
?
Valerate
–
?
?
Citrate
–
?
–
L-Proline
?
?
–
D-Ribose
?
–
–
Malonate
–
?
?
Acetate
?
–
–
5-Ketogluconate
?
?
–
Glycogen
–
?
–
API ZYM test results Trypsin
?
–
–
Acid phosphatase
–
?
?
G?C content (mol%)
68.0
71.9
69.3
T
Strains: 1, Roseomonas sediminicola FW-3 ; 2, Roseomonas lacus KACC 11678T; 3, Roseomonas terrae KACC 12677T. All data from this study, except the DNA G?C contents of the reference strains (taken from Jiang et al. 2006; Yoon et al. 2007). All strains are Gram-reaction-negative, positive for catalase, and oxidase activities but negative for hydrolysis of xylan, starch, Tween 80, tyrosine and casein. In API 20 NE and API ID 32 GN kits all strains are positive for 2-ketogluconate, 3-hydroxy-butyrate, suberate, and L-alanine activities. All strains are negative for reduction of nitrate to nitrogen, indole production, acid production from glucose, and assimilation of the following substrates: D-mannitol, D-glucose, salicin, Dmelibiose, L-fucose, D-sorbitol, caprate, L-histidine, 4-hydroxy-benzoate, Lrhamnose, N-acetyl-D-glucosamine, inositol, D-sucrose, D-maltose, itaconate, lactate, 3-hydroxy-benzoate, and L-serine. In API ZYM kits, all the strains are positive for alkaline phosphatase, esterase (C4), and esterase lipase (C8) activities but negative for lipase (C14), leucine arylamidase, valine arylamidase, crystine arylamidase, a-chymotrypsin, naphtol-AS-BIphosphohydrolase, a-galactosidase, b-galactosidase, b-glucuronidase, aglucosidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase, and a-fucosidase. ? Positive; – negative
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DNA G?C content and DNA–DNA hybridization The DNA G?C content of strain FW-3T was determined to be 68.0 mol%, a value comparable to those of R. lacus TH-G33T (71.9 %; Jiang et al. 2006) and R. terrae DS-48T (69.3 %; Yoon et al. 2007) and in the middle of range when compared to the other species in the genus Roseomonas (65.8–72.1 mol %). DNA– DNA relatedness values between strain FW-3T and R.
Antonie van Leeuwenhoek (2014) 105:191–197
195 Roseomonas lacus TH-G33T (AJ786000)
95 100
Roseomonas terrae DS-48T (EF363716)
Roseomonas sediminicola sp. nov. FW-3T (JQ349047) Roseomonas aquatica TR53T (AM231587) Roseomonas ludipueritiae 170/96T (AJ488504) Roseomonas aestuarii JC17T (FM244739)
100
Roseomonas cervicalis ATCC 49957T (AY150047) Roseomonas frigidaquae CW67T (EU290160) Roseomonas stagni HS-69T (AB369258)
100
96 100
Roseomonas gilardii subsp. gilardii ATCC 49956T (AY150045) Roseomonas gilardii subsp. rosea MDA5605T (AY220740) Roseomonas mucosa MDA5527T (AF538712) Roseomonas rosea 173/96T (AJ488505) Roseomonas pecuniae N75T (GU168019)
80
Roseomonas aerilata 5420S-30T (EF661571) 98
Roseomonas vinacea CPCC 100056T (EF368368) Craurococcus roseus NS130T (D85828) Paracraurococcus ruber NS89T (D85827)
84
Belnapia moabensis CP2CT (AJ871428) Roseococcus suduntuyensis SHETT (EU012448) Rubritepida flocculans DSM 14296T (AF465832)
100
Rhodovarius lipocyclicus CCUG 44693T (AJ633644) Acidisphaera rubrifaciens HS-AP3T (D86512)
98 69
Rhodovastum atsumiense G211T (AB381935) Acetobacter aceti NCIMB 8621T (X74066)
89
Gluconobacter cerinus IFO 3267T (X80775)
100 100
Gluconobacter oxydans NBRC 14819T (AB178433) Azospirillum brasilense ATCC 49958T (AY150046)
0.02
Fig. 1 Phylogenetic tree constructed from a comparative analysis of 16S rRNA gene sequences showing the relationships of Roseomonas sediminicola FW-3T with other related species. This tree was made using the neighbour-joining method (Saitou and Nei 1987) with a Kimura (1983) two-parameter distance
matrix and pairwise deletion. Dots indicate branches that were also recovered by using the maximum-parsimony algorithm. Bootstrap values (expressed as percentages of 1,000 replications) greater than 70 % are shown at the branch points. Bar 0.01 substitutions per 1 nucleotide position
lacus KACC 11678T and R. terrae KACC 12677T were 5 ± 1.2 and 4 ± 1.7 %, respectively. According to Wayne et al. (1987), DNA–DNA relatedness values lower than 70 % are considered to be the threshold value for the delineation of genospecies, so the result obtained is low enough to assign strain FW-3T as a novel species of the genus Roseomonas.
phylogenetic analysis warrant the assignment of strain FW-3T to the genus Roseomonas as the type strain of a novel species, for which the name Roseomonas sediminicola sp. nov. is proposed.
Taxonomic conclusions The characteristics of strain FW-3T are consistent with the description of the genus Roseomonas with regard to morphological, biochemical and chemotaxonomic properties. However, the phylogenetic distance between strain FW-3T and recognized Roseomonas species, the unique phenotypic characteristics (Table 1) and the low level of DNA–DNA relatedness values with the nearest neighbours identified by
Description of Roseomonas sediminicola sp. nov. Roseomonas sediminicola (se.di.mi.ni.co0 la. L. n. sedimen, -inis sediment; L. suff. -cola inhabitant, dweller; N.L. n. sediminicola sediment-dweller, referring to the source of the type strain). Cells are Gram-stain negative, strictly aerobic, nonmotile and non-spore-forming rods (0.3–0.5 lm in diameter and 1.5–2.2 lm in length) after culture on R2A agar for 2 days. Colonies are smooth, transparent, convex, circular with regular margins, pale red in colour, and 2–3 mm in diameter after 2 days on R2A agar. Growth also occurs on nutrient agar and TSA,
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Table 2 Cellular fatty acid profiles of strain FW-3T and phylogenetically related species of the genus Roseomonas Fatty acid
1
2
3
C12:0
1.5
–
–
C16:0
8.9
8.0
11.1
C16:1 x5c
0.9
2.2
2.6
C17:1 x7c
1.1
–
–
Saturated
Acknowledgments This work was supported by the Intelligent Synthetic Biology Center of Global Frontier Project funded by the Ministry of Education, Science and Technology (2011-0031967) and by the project on survey and excavation of Korean indigenous species of the National Institute of Biological Resources (NIBR) under the Ministry of Environment, Korea and by the China Major Projects No.2012ZX09503001-003.
Unsaturated
Hydroxy fatty acids C16:0 2OH
–
1.7
4.0
C16:0 3OH
–
1.5
4.3
C18:1 2OH
29.8
8.7
20.7
1.2
5.0
11.4
Cyclo fatty acid C19:0 cyclo x8c Summed feature 3; C14:0 3OH/iso-C16:1
1.3
–
–
4; C16:1 x7c/iso-C15:0 2OH 7; C18:1 x9c/x12t/x7c
– 55.4
4.6 68.2
– 45.9
Summed feature represent groups of two or three fatty acids that could not be separated by GLC with the MIDI system. Summed feature consist of: 3, C16:1 x7c/C16:1 x6c; 8, C18:1 x7c/x6c; 4, C16:1 x7c/iso-C15:0 2OH; 7, C18:1 x9c/x12t/x7c. Strains: 1, Roseomonas sediminicola FW-3T; 2, Roseomonas lacus KACC 11678T; 3, Roseomonas terrae KACC 12677T. All data from this study. All strains were cultured on nutrient agar for 2 days at 30 °C. Fatty acids amounting to \0.5 % of the total fatty acids in all strains are not listed. -, not detected. tr, trace amount below 0.5 %
but not on MacConkey. Grows on R2A at 10–37 °C and at pH 5.5–10, but not at 42 °C. Optimum growth occurs at 25–37 °C and at pH 7.0. Growth occurs well without NaCl supplement and in the presence of 1.0 % (w/v) NaCl. Catalase and oxidase are positive. Does not hydrolyze casein, DNA, xylan, skim milk, tyrosine, tween 80, and starch. Carbon assimilation tests as a sole carbon sources (API ID 32 GN, API 20 NE) and the enzyme activities (API ZYM) are listed in Table 1. Q-10 is the predominant respiratory quinone, and summed feature 7 (comprising C18:1 x9c/x12t/x7c) and C18:1 2OH are the major cellular fatty acids. The G?C content of the genomic DNA of the type strain is 68.0 mol %. The type strain, FW-3T (=KACC 16616T = JCM 18210T) was isolated from the fresh water of duck lake, KAIST, Daejeon, South Korea. The GenBank accession number of the 16S rRNA gene is JQ349047.
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