International Journal of Systematic and Evolutionary Microbiology (2014), 64, 3040–3045
DOI 10.1099/ijs.0.061945-0
Parafilimonas terrae gen. nov., sp. nov., isolated from greenhouse soil Soo-Jin Kim,1 Joo-Hyeon Park,1 Jun-Muk Lim,1,2 Jae-Hyung Ahn,1 Rangasamy Anandham,3 Hang-Yeon Weon1 and Soon-Wo Kwon1 Correspondence Soon-Wo Kwon [email protected]
1
Agricultural Microbiology Division, National Academy of Agricultural Science, Rural Development Administration, Suwon 441-707, Republic of Korea
2
Department of Microbiology, Dankook University, Anseo-dong, Cheonan 330-714, Republic of Korea
3
Department of Agricultural Microbiology, Agricultural College and Research Institute, Tamil Nadu Agricultural University, Madurai 625 104, Tamil Nadu, India
A Gram-stain-negative, short rod-shaped, non-flagellated, yellow bacterium, designated strain 5GHs7-2T, was isolated from a greenhouse soil sample in South Korea. 16S rRNA gene sequence analysis of strain 5GHs7-2T indicated that the isolate belonged to the family Chitinophagaceae, and exhibited the highest sequence similarities with members of the genera Terrimonas (89.2–92.6 %), Sediminibacterium (90.8–91.4 %) and Chitinophaga (89.2–91.7 %), Filimonas lacunae YT21T (91.7 %), members of the genus Segetibacter (90.2–91.6 %), Parasegetibacter luojiensis RHYL-37T (90.9 %) and Flavihumibacter petaseus T41T (91.2 %). Flexirubin-type pigments were present. The major cellular fatty acids of the novel strain were isoC15 : 0, iso-C17 : 0 3-OH and iso-C15 : 1 G. The polar lipid profile consisted of a large amount of phosphatidylethanolamine, and moderate and small amounts of several unknown aminolipids and lipids. The only respiratory quinone of strain 5GHs7-2T was MK-7, and the DNA G+C content was 47.6 mol%. On the basis of the evidence presented, it is concluded that strain 5GHs7-2T represents a novel species of a new genus in the family Chitinophagaceae, for which the name Parafilimonas terrae gen. nov., sp. nov. is proposed. The type strain of the type species is 5GHs72T (5KACC 17343T5DSM 28286T).
The family Chitinophagaceae, a member of the phylum Bacteroidetes, was proposed by Ka¨mpfer et al. (2011). Members of the family are aerobic or facultatively anaerobic, and usually non-motile. Cells are often thin rod-shaped, and contain the menaquinone type of MK-7, and iso-C15 : 0, isoC17 : 0 3-OH and iso-C15 : 1 G as the major fatty acids. At the time of writing, the family Chitinophagaceae includes 16 genera including the type genus Chitinophaga. Strain 5GHs7-2T was isolated from a greenhouse soil sample in Yongjin-myeon, Wanju-gun, Republic of Korea. Isolation and subcultivation was conducted on R2A medium (BD). Cell morphology was examined with an electron microscope (912AB; LEO) using cells grown on R2A agar at 28 uC for 2 days, followed by negative staining with 1 % Abbreviation: PE, phosphatidylethanolamine. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain 5GHs7-2T is KF934397. Two supplementary figures are available with the online version of this paper.
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(w/v) sodium phosphotungstic acid (pH 7.0). Growth of strain 5GHs7-2T was assessed in R2A at 5–45 uC (at 5 uC intervals) and pH 4.0–12.0 (at 1.0 pH unit intervals). R2A broth was prepared using citrate/phosphate buffer, Trishydrochloride buffer, HCl or NaOH (Breznak & Costilow, 1994). Growth in the presence of 0–5 % (w/v) NaCl (at 1 % intervals) was investigated using R2A broth. Gram staining was checked using a bioMe´rieux Gram stain kit according to the manufacturer’s instructions. Casein, CM-cellulose, hypoxanthine, starch, Tween 80, tyrosine and xanthine degradations were examined on R2A plates containing milk powder (5 %, w/v), CM-cellulose (1 %, w/v), hypoxanthine (0.5 %, w/v), starch (1 %, w/v), Tween 80 (1 %, w/v), tyrosine (0.1 %, w/v) and xanthine (0.5 %, w/v), respectively. DNase activity was determined with DNase test agar (Difco). The KOH test was used to detect flexirubin pigments (Bernardet et al., 2002). Oxidase activity was tested by the oxidation of 1 % (w/v) tetramethyl p-phenylenediamine and catalase activity was evaluated by the production of oxygen bubbles in 3 % (v/v) aqueous hydrogen peroxide solution. Additional enzyme activities, biochemical features and the utilization of carbon sources were determined by using 061945 G 2014 IUMS Printed in Great Britain
http://ijs.sgmjournals.org
Table 1. Characteristics that differentiate strain 5GHs7-2T from other members of the family Chitinophagaceae Strains: 1, 5GHs7-2T; 2, Chitinophaga arvensicola IAM 12650T (data from Han et al., 2014; Oyaizu et al., 1982; Ka¨mpfer et al., 2006); 3, Chitinophaga polysaccharea MRP-15T (Han et al., 2014); 4, Filimonas lacunae YT21T (Shiratori et al., 2009); 5, Flavihumibacter petaseus T41T (Zhang et al., 2010); 6, Terrimonas lutea DYT (Jin et al., 2013; Xie & Yokota, 2006); 7, Terrimonas pekingensis QHT (Jin et al., 2013); 8, Segetibacter aerophilus 6424S-61T (Weon et al., 2010). +, Positive; W, weak positive; 2, negative; ND, not available. Characteristic
1
2
4
Rhizoplane Yellow 0.3–0.460.6–0.9 Rod 2
4–45 + 4.0–10.0 2.0
Fresh water Light yellow 0.3–0.565.0–30.0 Filamentous + 2 + + 2 + + + 10–35 2 5.0–9.0 1.0
47.9
45.2
ND
2 2 + + 2 ND
5
6
Soil Soil Yellow Yellow 0.4–0.560.5–0.6 0.3–0.561.0–3.0 Rod Rod 2 2 ND 2 W + W + 2 + ND + + + 2 + 20–37 10–37 + + ND 5.5–9.5 1.0 1.0 48.1
47.2
7
8
Sludge Air White Yellow 0.3-0.561.0-3.0 0.9–1.163.0–6.3 Rod Rod 2 2 2 ND + 2 + 2 W 2 + + 2 + + 10–37 15–30 + 2 5.0–8.0 ND 1.0 1.0 41.0
38.4
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Parafilimonas terrae gen. nov., sp. nov.
Isolation source Soil Soil Pigmentation Yellow Yellow-orange 0.65–0.7561.2–2.0 0.360.8–2.0 Cell width 6 length (mm) Morphology Short rod Rod Motility 2 2 Flexirubin production + + Oxidase 2 + Catalase + + Nitrate reduction 2 + Aesculin + + Gelatin liquefaction + 2 DNase + + Temperature range for growth (uC) 15–40 5–37 Growth at 37 uC + + pH range for growth 5.0–8.0 4.5–8.0 Maximum NaCl concentration for 1.0 2.0 growth (%, w/v) DNA G+C content (mol%) 47.6 46.0
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S.-J. Kim and others
API ZYM, API 20NE and API ID 32GN kits (bioMe´rieux). Strain 5GHs7-2T was Gram-stain-negative, aerobic, nonflagellated and short rod-shaped (Fig. S1, available in the online Supplementary Material). It was catalase-positive and oxidase-negative, and grew between 15 uC and 40 uC and between pH 5.0 and pH 8.0. Phenotypic comparisons of strain 5GHs7-2T and closely related taxa are shown in Table 1. The 16S rRNA gene of strain 5GHs7-2T was amplified by PCR using the universal primers 9F and 1512R (Weisburg et al., 1991). The purified PCR products were sequenced by Genotech Inc., Daejeon, South Korea. The sequence obtained was compared with closely related sequences of reference organisms in the Eztaxon-e database using the Eztaxon-e identifying function (Kim et al., 2012; http:// eztaxon-e.ezbiocloud.net/). Calculation of sequence similarity levels were carried out by using the EzTaxon-e server. Multiple alignments with sequences of a broad selection of members of the family Chitinophagaceae were conducted using ARB software (Ludwig et al., 2004), and were added to the alignment of the SILVA SSURef version 106 ARB database (Pruesse et al., 2007). Phylogenetic trees were reconstructed using MEGA software version 5.0 (Tamura et al., 2011) with the neighbour-joining (Saitou & Nei, 1987), maximumparsimony (Kluge & Farris, 1969) and maximum-likelihood
99 ●
0.02 ○
100●
(Felsenstein, 1981) algorithms. The nearly complete 16S rRNA gene sequence of strain 5GHs7-2T (1461 bp) was determined. Strain 5GHs7-2T showed the highest16S rRNA gene sequence similarities with members of the genera Terrimonas (89.2–92.6 %), Sediminibacterium (90.8–91.4 %) and Chitinophaga (89.2–91.7 %), Filimonas lacunae YT21T (91.7 %), members of the genus Segetibacter (90.2–91.6 %), Parasegetibacter luojiensis RHYL-37T (90.9 %) and Flavihumibacter petaseus T41T (91.2 %). According to the neighbourjoining phylogenetic tree, strain 5GHs7-2T grouped with the genera Filimonas, Flavihumibacter, Chitinophaga, Parasegetibacter and Segetibacter within the family Chitinophagaceae (Fig. 1). This cluster was also generated in the maximumlikelihood tree although not in the maximum-parsimony tree (Fig. 1). Analyses of isoprenoid quinones and polar lipids were conducted with freeze-dried cells after cultivation in R2A broth for 2 days at 28 uC. Isoprenoid quinones were analysed by HPLC as described by Groth et al. (1996). Polar lipids were extracted and then examined by twodimensional TLC (Minnikin et al., 1984). For detection of polar lipids, plates were sprayed with molybdatophosphoric acid (for total lipids), phosphomolybdic acid (for phospholipids), ninhydrin (for aminolipids) and
Chitinophaga polysaccharea MRP-15T (KC430923) Chitinophaga arvensicola DSM 3695T (AM237311) Chitinophaga pinensis DSM 2588T (CP001699) Flavihumibacter petaseus T41T (EU854577)
○
Filimonas lacunae YT21T (AB362776)
○
Parafilimonas terrae 5GHs7-2T (KF934397) Parasegetibacter luojiensis RHYL-37T (EU877263)
●
Segetibacter koreensis DSM 18137T (ARFB01000010)
● 99
Segetibacter aerophilus 6424S-61T (GQ421847) 100● Niastella yeongjuensis GR20-13T (DQ244076) Niastella koreensis GR20-10T (CP003178) Flavitalea populi HY-50RT (HM130561)
○ 79○
Flavisolibacter ginsengiterrae Gsoil 492T (AB267476)
100● ● ● 75
100● ○
Terrimonas pekingensis QHT (JF834159) Terrimonas lutea DYT (AB192292) Terrimonas ferruginea DSM 30193T (AM230484) Niabella aurantiaca R2A15-11T (DQ457019) Ferruginibacter alkalilentus HU1-GD23T (FJ177530) Sediminibacterium salmoneum NJ-44T (EF407879)
● 98
Hydrotalea flava CCUG 51397T (FN665659) Lacibacter cauensis NJ-8T (EU521690)
●
Flavobacterium aquatile ATCC 11947T (M62797) ● 100
Gracilimonas tropica CL-CB462T (EF988655) Balneola vulgaris 13IX/A01/164T (AY576749)
Fig. 1. Neighbour-joining tree, based on 16S rRNA gene sequences, showing the phylogenetic relationships of strain 5GHs72T and some other related taxa. Circles indicate nodes identical to those seen in the corresponding maximum-likelihood tree (open circles), or both the maximum-likelihood and maximum-parsimony trees (filled circles). Numbers at nodes are percentage bootstrap values (based on 1000 replications); only values .70 % are shown. Bar, 0.02 substitutions per nucleotide position. 3042
International Journal of Systematic and Evolutionary Microbiology 64
Parafilimonas terrae gen. nov., sp. nov.
a-naphthol/sulfuric acid reagent (for glycolipids). Cellular
fatty acid methyl esters of strain 5GHs7-2T were extracted from exponential growth phase cells grown on R2A agar for 2 days at 28 uC, and were analysed by GC according to the instructions of the Microbial Identification System (MIDI). The fatty acid methyl esters were identified and quantified by using the TSBA 6 database (version 6.10) of the Sherlock Microbial Identification System (MIDI). DNA 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). The polar lipid profile of strain 5GHs7-2T contained phosphatidylethanolamine (PE), five unknown aminolipids (ALs) and seven unknown lipids (Ls) (Fig. S2). The polar lipid profiles of strain 5GHs7-2T and the closely related genera Filimonas, Chitinophaga and Terrimonas had PE in common, but showed differences in other polar lipids such as unknown aminolipids and unknown lipids (Chung et al., 2012; Jin et al., 2013; Leandro et al., 2013). The only respiratory quinone of strain 5GHs7-2T was MK-7, in line with those of other members of the family Chitinophagaceae (Ka¨mpfer et al., 2011). The cellular fatty acids of strain 5GHs7-2T were iso-C15 : 0 (37.6 %), iso-C17 : 0 3-OH (20.7 %), iso-C15 : 1 G (17.3 %), C16 : 0 (4.7 %), C17 : 0 2-OH (3.6 %), C16 : 0 3-OH (3.2 %), iso-C15 : 0 3-OH (2.9 %), anteiso- C15 : 0 (2.3 %), summed feature 3 (C16 : 1v7c and/or C16 : 1v6c; 2.1 %), C14 : 0 (1.7 %), anteiso-C15 : 1 A (1.5 %), iso-C16 : 0 3OH (1.2 %), isoC16 : 0 (1.0 %) and iso-C13 : 0 (0.5 %). The differences in the fatty acids between strain 5GHs7-2T and members of closely related genera are shown in Table 2. The DNA G+C content of strain 5GHs7-2T was 47.6 mol%. In conclusion, the relatively low 16S rRNA gene sequence similarity values between strain 5GHs7-2T and members of related genera of the family Chitinophagaceae, together with phenotypic, phylogenetic and chemotaxonomic data, demonstrates that strain 5GHs7-2T belongs to the family Chitinophagaceae, and represents a novel species of a new genus, for which the name Parafilimonas terrae gen. nov., sp. nov., is proposed.
Table 2. Cellular fatty acid composition (%) of strain 5GHs72T and related strains Strains: 1, 5GHs7-2T; 2, Chitinophaga arvensicola IAM 12650T (data from Ka¨mpfer et al., 2006); 3, Chitinophaga polysaccharea MRP-15T (Han et al., 2014); 4, Filimonas lacunae YT21T (Shiratori et al., 2009); 5, Flavihumibacter petaseus T41T (Zhang et al., 2010); 6, Terrimonas lutea DYT (Jin et al., 2013); 7, Terrimonas pekingensis QHT (Jin et al., 2013); 8, Segetibacter aerophilus 6424S-61T (Weon et al., 2010). 2, Not detected or ,1.0 %. Fatty acid iso-C13 : 0 iso-C13 : 0 3-OH C14 : 0 C15 : 0 anteiso-C15 : 0 anteiso-C15 : 1 A iso-C15 : 0 iso-C15 : 0 3-OH iso-C15 : 1 G C16 : 0 C16 : 0 2-OH C16 : 0 3-OH C16 : 0 N alcohol C16 : 1v5c iso-C16 : 0 iso-C16 : 0 3-OH C17 : 0 2-OH C17 : 0 3-OH C17 : 1v6c iso-C17 : 0 iso-C17 : 0 3-OH Unknown ECL11.543 ECL13.565 ECL14.959 ECL16.582 Summed feature* 3 4
1
2
3
4
5
6
7
8
2 2 2 2 2 2 2 3.1 2 2 2 2 2 2 2 2.1 1.7 1.4 1.7 2 2 2 1.6 2 2 2 2 2 8.0 2 2 2 2.3 2 2.2 2 2 2 3.1 4.7 1.5 2 2 2 2 2 2 2 37.6 35.3 37.7 25.2 33.8 19.0 27.8 16.9 2.9 3.0 2.0 2.7 3.9 2.4 1.6 2 17.3 2 2 18.3 13.3 18.6 11.7 26.4 4.7 5.1 5.5 4.7 2.2 1.9 8.2 5.0 2 2 2 2 2 2 1.7 2 3.2 1.7 2 3.9 2.7 7.4 5.2 2 2 2 2 2 2 2 4.1 2 2 33.6 34.1 3.0 5.8 2 2 9.8 1.0 2 1.1 2 2 2 2 1.1 1.2 2 2 1.0 2 2 1.4 2 3.6 2 2 2 2 2 2 3.2 2 2 2 2 1.1 3.1 2.4 2 2 2 2 2 2 2 1.3 1.2 2 2 2 2 2 2 2 1.6 20.7 5.9 1.4 22.6 12.9 15.1 8.9 7.5 2 2 2 2
2 3.6 2 2
1.8 4.6 2 2
2.1 2
3.7 2
2.6 13.6 2 2
2 2 2 1.9
2 1.5 2 2
2 2 2 2
2 2 2 2
8.3 26.6 14.6 2 2 5.5
2 2 2 2
8.8 2.6
Description of Parafilimonas gen. nov. Parafilimonas (Pa.ra.fi.li.mo9nas. Gr. prep. para beside; N.L. fem. n. Filimonas the name of a bacterial genus; N.L. fem. n. Parafilimonas resembling the genus Filimonas). Gram-stain-negative, aerobic, non-flagellated, catalase-positive and oxidase-negative. Cells are short rods. The only menaquinone is MK-7. The polar lipids comprise a large amount of PE, and moderate and small amounts of five unknown aminolipids and seven unknown lipids. The predominant cellular fatty acids are iso-C15 : 0, iso-C17 : 0 3-OH and iso-C15 : 1 G. A member of the family Chitinophagaceae. The type species is Parafilimonas terrae. Description of Parafilimonas terrae sp. nov. Parafilimonas terrae (ter9rae. L. gen. n. terrae of/from soil). http://ijs.sgmjournals.org
*Summed features represent groups of two or more fatty acids that could not be separated by GC with the MIDI system. Summed feature 3 consisted of one or more of C16 : 1v7c, C16 : 1v6c and iso-C15 : 0 2-OH; summed feature 4 consisted of anteiso-C17 : 1 B and/or iso-C17 : 1 I.
Displays the following properties in addition to those given in the genus description. Cells are approximately 0.65– 0.75 mm61.2–2.0 mm. Colonies are irregular and yellow on R2A. Growth occurs at 15–40 uC (optimum 28–30 uC) and pH 5.0–8.0 (optimum, pH 7.0). Grows in the presence of 0–1 % (w/v) NaCl (optimum 1 %). Hydrolyses casein, DNA, Tween 80 and tyrosine, but not CM-cellulose, hypoxanthine, starch or xanthine. Produces flexirubin pigment. Positive reactions for aesculin hydrolysis and gelatin hydrolysis, but negative results for nitrate reduction, indole 3043
S.-J. Kim and others
production, glucose fermentation, arginine dihydrolase and urease (API 20NE test strips). Assimilate D-glucose, Larabinose, D-mannose, N-acetylglucosamine, maltose, sucrose, glycogen, salicin and melibiose, but not D-mannitol, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate, phenylacetic acid, L-rhamnose, D-ribose, inositol, itaconic acid, suberic acid, sodium malonate, sodium acetate, lactic acid, L-alanine, potassium 5-ketogluconate, 3-hydroxybenzoic acid, L-serine, L-fucose, D-sorbitol, propionic acid, valeric acid, L-histidine, potassium 2ketogluconate, 3-hydroxybutyric acid, 4-hydroxybenzoic acid or L-proline (API 20NE and API ID 32GN test strips). Positive results for activities of alkaline phosphatase, leucine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, a-galactosidase, b-galactosidase, N-acetyl-b-glucosaminidase and a-fucosidase, but negative results for esterase (C4), esterase lipase (C8), lipase (C14), valine arylamidase, cystine arylamidase, trypsin, a-chymotrypsin, b-glucuronidase, a-glucosidase, b-glucosidase and a-mannosidase activities (API ZYM test strips). The type strain is 5GHs7-2T (5KACC 17343T5DSM 28286T), isolated from greenhouse soil at Yongjin–myeon, Wanju-gun, Republic of Korea. The DNA G+C content of the type strain is 47.6 mol%.
Acknowledgements This study was carried out with the support of the National Academy of Agricultural Science, Rural Development Administration, Republic of Korea (project no. PJ008666).
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DOI 10.1099/ijs.0.061945-0
Parafilimonas terrae gen. nov., sp. nov., isolated from greenhouse soil Soo-Jin Kim,1 Joo-Hyeon Park,1 Jun-Muk Lim,1,2 Jae-Hyung Ahn,1 Rangasamy Anandham,3 Hang-Yeon Weon1 and Soon-Wo Kwon1 Correspondence Soon-Wo Kwon [email protected]
1
Agricultural Microbiology Division, National Academy of Agricultural Science, Rural Development Administration, Suwon 441-707, Republic of Korea
2
Department of Microbiology, Dankook University, Anseo-dong, Cheonan 330-714, Republic of Korea
3
Department of Agricultural Microbiology, Agricultural College and Research Institute, Tamil Nadu Agricultural University, Madurai 625 104, Tamil Nadu, India
A Gram-stain-negative, short rod-shaped, non-flagellated, yellow bacterium, designated strain 5GHs7-2T, was isolated from a greenhouse soil sample in South Korea. 16S rRNA gene sequence analysis of strain 5GHs7-2T indicated that the isolate belonged to the family Chitinophagaceae, and exhibited the highest sequence similarities with members of the genera Terrimonas (89.2–92.6 %), Sediminibacterium (90.8–91.4 %) and Chitinophaga (89.2–91.7 %), Filimonas lacunae YT21T (91.7 %), members of the genus Segetibacter (90.2–91.6 %), Parasegetibacter luojiensis RHYL-37T (90.9 %) and Flavihumibacter petaseus T41T (91.2 %). Flexirubin-type pigments were present. The major cellular fatty acids of the novel strain were isoC15 : 0, iso-C17 : 0 3-OH and iso-C15 : 1 G. The polar lipid profile consisted of a large amount of phosphatidylethanolamine, and moderate and small amounts of several unknown aminolipids and lipids. The only respiratory quinone of strain 5GHs7-2T was MK-7, and the DNA G+C content was 47.6 mol%. On the basis of the evidence presented, it is concluded that strain 5GHs7-2T represents a novel species of a new genus in the family Chitinophagaceae, for which the name Parafilimonas terrae gen. nov., sp. nov. is proposed. The type strain of the type species is 5GHs72T (5KACC 17343T5DSM 28286T).
The family Chitinophagaceae, a member of the phylum Bacteroidetes, was proposed by Ka¨mpfer et al. (2011). Members of the family are aerobic or facultatively anaerobic, and usually non-motile. Cells are often thin rod-shaped, and contain the menaquinone type of MK-7, and iso-C15 : 0, isoC17 : 0 3-OH and iso-C15 : 1 G as the major fatty acids. At the time of writing, the family Chitinophagaceae includes 16 genera including the type genus Chitinophaga. Strain 5GHs7-2T was isolated from a greenhouse soil sample in Yongjin-myeon, Wanju-gun, Republic of Korea. Isolation and subcultivation was conducted on R2A medium (BD). Cell morphology was examined with an electron microscope (912AB; LEO) using cells grown on R2A agar at 28 uC for 2 days, followed by negative staining with 1 % Abbreviation: PE, phosphatidylethanolamine. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain 5GHs7-2T is KF934397. Two supplementary figures are available with the online version of this paper.
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(w/v) sodium phosphotungstic acid (pH 7.0). Growth of strain 5GHs7-2T was assessed in R2A at 5–45 uC (at 5 uC intervals) and pH 4.0–12.0 (at 1.0 pH unit intervals). R2A broth was prepared using citrate/phosphate buffer, Trishydrochloride buffer, HCl or NaOH (Breznak & Costilow, 1994). Growth in the presence of 0–5 % (w/v) NaCl (at 1 % intervals) was investigated using R2A broth. Gram staining was checked using a bioMe´rieux Gram stain kit according to the manufacturer’s instructions. Casein, CM-cellulose, hypoxanthine, starch, Tween 80, tyrosine and xanthine degradations were examined on R2A plates containing milk powder (5 %, w/v), CM-cellulose (1 %, w/v), hypoxanthine (0.5 %, w/v), starch (1 %, w/v), Tween 80 (1 %, w/v), tyrosine (0.1 %, w/v) and xanthine (0.5 %, w/v), respectively. DNase activity was determined with DNase test agar (Difco). The KOH test was used to detect flexirubin pigments (Bernardet et al., 2002). Oxidase activity was tested by the oxidation of 1 % (w/v) tetramethyl p-phenylenediamine and catalase activity was evaluated by the production of oxygen bubbles in 3 % (v/v) aqueous hydrogen peroxide solution. Additional enzyme activities, biochemical features and the utilization of carbon sources were determined by using 061945 G 2014 IUMS Printed in Great Britain
http://ijs.sgmjournals.org
Table 1. Characteristics that differentiate strain 5GHs7-2T from other members of the family Chitinophagaceae Strains: 1, 5GHs7-2T; 2, Chitinophaga arvensicola IAM 12650T (data from Han et al., 2014; Oyaizu et al., 1982; Ka¨mpfer et al., 2006); 3, Chitinophaga polysaccharea MRP-15T (Han et al., 2014); 4, Filimonas lacunae YT21T (Shiratori et al., 2009); 5, Flavihumibacter petaseus T41T (Zhang et al., 2010); 6, Terrimonas lutea DYT (Jin et al., 2013; Xie & Yokota, 2006); 7, Terrimonas pekingensis QHT (Jin et al., 2013); 8, Segetibacter aerophilus 6424S-61T (Weon et al., 2010). +, Positive; W, weak positive; 2, negative; ND, not available. Characteristic
1
2
4
Rhizoplane Yellow 0.3–0.460.6–0.9 Rod 2
4–45 + 4.0–10.0 2.0
Fresh water Light yellow 0.3–0.565.0–30.0 Filamentous + 2 + + 2 + + + 10–35 2 5.0–9.0 1.0
47.9
45.2
ND
2 2 + + 2 ND
5
6
Soil Soil Yellow Yellow 0.4–0.560.5–0.6 0.3–0.561.0–3.0 Rod Rod 2 2 ND 2 W + W + 2 + ND + + + 2 + 20–37 10–37 + + ND 5.5–9.5 1.0 1.0 48.1
47.2
7
8
Sludge Air White Yellow 0.3-0.561.0-3.0 0.9–1.163.0–6.3 Rod Rod 2 2 2 ND + 2 + 2 W 2 + + 2 + + 10–37 15–30 + 2 5.0–8.0 ND 1.0 1.0 41.0
38.4
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Parafilimonas terrae gen. nov., sp. nov.
Isolation source Soil Soil Pigmentation Yellow Yellow-orange 0.65–0.7561.2–2.0 0.360.8–2.0 Cell width 6 length (mm) Morphology Short rod Rod Motility 2 2 Flexirubin production + + Oxidase 2 + Catalase + + Nitrate reduction 2 + Aesculin + + Gelatin liquefaction + 2 DNase + + Temperature range for growth (uC) 15–40 5–37 Growth at 37 uC + + pH range for growth 5.0–8.0 4.5–8.0 Maximum NaCl concentration for 1.0 2.0 growth (%, w/v) DNA G+C content (mol%) 47.6 46.0
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S.-J. Kim and others
API ZYM, API 20NE and API ID 32GN kits (bioMe´rieux). Strain 5GHs7-2T was Gram-stain-negative, aerobic, nonflagellated and short rod-shaped (Fig. S1, available in the online Supplementary Material). It was catalase-positive and oxidase-negative, and grew between 15 uC and 40 uC and between pH 5.0 and pH 8.0. Phenotypic comparisons of strain 5GHs7-2T and closely related taxa are shown in Table 1. The 16S rRNA gene of strain 5GHs7-2T was amplified by PCR using the universal primers 9F and 1512R (Weisburg et al., 1991). The purified PCR products were sequenced by Genotech Inc., Daejeon, South Korea. The sequence obtained was compared with closely related sequences of reference organisms in the Eztaxon-e database using the Eztaxon-e identifying function (Kim et al., 2012; http:// eztaxon-e.ezbiocloud.net/). Calculation of sequence similarity levels were carried out by using the EzTaxon-e server. Multiple alignments with sequences of a broad selection of members of the family Chitinophagaceae were conducted using ARB software (Ludwig et al., 2004), and were added to the alignment of the SILVA SSURef version 106 ARB database (Pruesse et al., 2007). Phylogenetic trees were reconstructed using MEGA software version 5.0 (Tamura et al., 2011) with the neighbour-joining (Saitou & Nei, 1987), maximumparsimony (Kluge & Farris, 1969) and maximum-likelihood
99 ●
0.02 ○
100●
(Felsenstein, 1981) algorithms. The nearly complete 16S rRNA gene sequence of strain 5GHs7-2T (1461 bp) was determined. Strain 5GHs7-2T showed the highest16S rRNA gene sequence similarities with members of the genera Terrimonas (89.2–92.6 %), Sediminibacterium (90.8–91.4 %) and Chitinophaga (89.2–91.7 %), Filimonas lacunae YT21T (91.7 %), members of the genus Segetibacter (90.2–91.6 %), Parasegetibacter luojiensis RHYL-37T (90.9 %) and Flavihumibacter petaseus T41T (91.2 %). According to the neighbourjoining phylogenetic tree, strain 5GHs7-2T grouped with the genera Filimonas, Flavihumibacter, Chitinophaga, Parasegetibacter and Segetibacter within the family Chitinophagaceae (Fig. 1). This cluster was also generated in the maximumlikelihood tree although not in the maximum-parsimony tree (Fig. 1). Analyses of isoprenoid quinones and polar lipids were conducted with freeze-dried cells after cultivation in R2A broth for 2 days at 28 uC. Isoprenoid quinones were analysed by HPLC as described by Groth et al. (1996). Polar lipids were extracted and then examined by twodimensional TLC (Minnikin et al., 1984). For detection of polar lipids, plates were sprayed with molybdatophosphoric acid (for total lipids), phosphomolybdic acid (for phospholipids), ninhydrin (for aminolipids) and
Chitinophaga polysaccharea MRP-15T (KC430923) Chitinophaga arvensicola DSM 3695T (AM237311) Chitinophaga pinensis DSM 2588T (CP001699) Flavihumibacter petaseus T41T (EU854577)
○
Filimonas lacunae YT21T (AB362776)
○
Parafilimonas terrae 5GHs7-2T (KF934397) Parasegetibacter luojiensis RHYL-37T (EU877263)
●
Segetibacter koreensis DSM 18137T (ARFB01000010)
● 99
Segetibacter aerophilus 6424S-61T (GQ421847) 100● Niastella yeongjuensis GR20-13T (DQ244076) Niastella koreensis GR20-10T (CP003178) Flavitalea populi HY-50RT (HM130561)
○ 79○
Flavisolibacter ginsengiterrae Gsoil 492T (AB267476)
100● ● ● 75
100● ○
Terrimonas pekingensis QHT (JF834159) Terrimonas lutea DYT (AB192292) Terrimonas ferruginea DSM 30193T (AM230484) Niabella aurantiaca R2A15-11T (DQ457019) Ferruginibacter alkalilentus HU1-GD23T (FJ177530) Sediminibacterium salmoneum NJ-44T (EF407879)
● 98
Hydrotalea flava CCUG 51397T (FN665659) Lacibacter cauensis NJ-8T (EU521690)
●
Flavobacterium aquatile ATCC 11947T (M62797) ● 100
Gracilimonas tropica CL-CB462T (EF988655) Balneola vulgaris 13IX/A01/164T (AY576749)
Fig. 1. Neighbour-joining tree, based on 16S rRNA gene sequences, showing the phylogenetic relationships of strain 5GHs72T and some other related taxa. Circles indicate nodes identical to those seen in the corresponding maximum-likelihood tree (open circles), or both the maximum-likelihood and maximum-parsimony trees (filled circles). Numbers at nodes are percentage bootstrap values (based on 1000 replications); only values .70 % are shown. Bar, 0.02 substitutions per nucleotide position. 3042
International Journal of Systematic and Evolutionary Microbiology 64
Parafilimonas terrae gen. nov., sp. nov.
a-naphthol/sulfuric acid reagent (for glycolipids). Cellular
fatty acid methyl esters of strain 5GHs7-2T were extracted from exponential growth phase cells grown on R2A agar for 2 days at 28 uC, and were analysed by GC according to the instructions of the Microbial Identification System (MIDI). The fatty acid methyl esters were identified and quantified by using the TSBA 6 database (version 6.10) of the Sherlock Microbial Identification System (MIDI). DNA 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). The polar lipid profile of strain 5GHs7-2T contained phosphatidylethanolamine (PE), five unknown aminolipids (ALs) and seven unknown lipids (Ls) (Fig. S2). The polar lipid profiles of strain 5GHs7-2T and the closely related genera Filimonas, Chitinophaga and Terrimonas had PE in common, but showed differences in other polar lipids such as unknown aminolipids and unknown lipids (Chung et al., 2012; Jin et al., 2013; Leandro et al., 2013). The only respiratory quinone of strain 5GHs7-2T was MK-7, in line with those of other members of the family Chitinophagaceae (Ka¨mpfer et al., 2011). The cellular fatty acids of strain 5GHs7-2T were iso-C15 : 0 (37.6 %), iso-C17 : 0 3-OH (20.7 %), iso-C15 : 1 G (17.3 %), C16 : 0 (4.7 %), C17 : 0 2-OH (3.6 %), C16 : 0 3-OH (3.2 %), iso-C15 : 0 3-OH (2.9 %), anteiso- C15 : 0 (2.3 %), summed feature 3 (C16 : 1v7c and/or C16 : 1v6c; 2.1 %), C14 : 0 (1.7 %), anteiso-C15 : 1 A (1.5 %), iso-C16 : 0 3OH (1.2 %), isoC16 : 0 (1.0 %) and iso-C13 : 0 (0.5 %). The differences in the fatty acids between strain 5GHs7-2T and members of closely related genera are shown in Table 2. The DNA G+C content of strain 5GHs7-2T was 47.6 mol%. In conclusion, the relatively low 16S rRNA gene sequence similarity values between strain 5GHs7-2T and members of related genera of the family Chitinophagaceae, together with phenotypic, phylogenetic and chemotaxonomic data, demonstrates that strain 5GHs7-2T belongs to the family Chitinophagaceae, and represents a novel species of a new genus, for which the name Parafilimonas terrae gen. nov., sp. nov., is proposed.
Table 2. Cellular fatty acid composition (%) of strain 5GHs72T and related strains Strains: 1, 5GHs7-2T; 2, Chitinophaga arvensicola IAM 12650T (data from Ka¨mpfer et al., 2006); 3, Chitinophaga polysaccharea MRP-15T (Han et al., 2014); 4, Filimonas lacunae YT21T (Shiratori et al., 2009); 5, Flavihumibacter petaseus T41T (Zhang et al., 2010); 6, Terrimonas lutea DYT (Jin et al., 2013); 7, Terrimonas pekingensis QHT (Jin et al., 2013); 8, Segetibacter aerophilus 6424S-61T (Weon et al., 2010). 2, Not detected or ,1.0 %. Fatty acid iso-C13 : 0 iso-C13 : 0 3-OH C14 : 0 C15 : 0 anteiso-C15 : 0 anteiso-C15 : 1 A iso-C15 : 0 iso-C15 : 0 3-OH iso-C15 : 1 G C16 : 0 C16 : 0 2-OH C16 : 0 3-OH C16 : 0 N alcohol C16 : 1v5c iso-C16 : 0 iso-C16 : 0 3-OH C17 : 0 2-OH C17 : 0 3-OH C17 : 1v6c iso-C17 : 0 iso-C17 : 0 3-OH Unknown ECL11.543 ECL13.565 ECL14.959 ECL16.582 Summed feature* 3 4
1
2
3
4
5
6
7
8
2 2 2 2 2 2 2 3.1 2 2 2 2 2 2 2 2.1 1.7 1.4 1.7 2 2 2 1.6 2 2 2 2 2 8.0 2 2 2 2.3 2 2.2 2 2 2 3.1 4.7 1.5 2 2 2 2 2 2 2 37.6 35.3 37.7 25.2 33.8 19.0 27.8 16.9 2.9 3.0 2.0 2.7 3.9 2.4 1.6 2 17.3 2 2 18.3 13.3 18.6 11.7 26.4 4.7 5.1 5.5 4.7 2.2 1.9 8.2 5.0 2 2 2 2 2 2 1.7 2 3.2 1.7 2 3.9 2.7 7.4 5.2 2 2 2 2 2 2 2 4.1 2 2 33.6 34.1 3.0 5.8 2 2 9.8 1.0 2 1.1 2 2 2 2 1.1 1.2 2 2 1.0 2 2 1.4 2 3.6 2 2 2 2 2 2 3.2 2 2 2 2 1.1 3.1 2.4 2 2 2 2 2 2 2 1.3 1.2 2 2 2 2 2 2 2 1.6 20.7 5.9 1.4 22.6 12.9 15.1 8.9 7.5 2 2 2 2
2 3.6 2 2
1.8 4.6 2 2
2.1 2
3.7 2
2.6 13.6 2 2
2 2 2 1.9
2 1.5 2 2
2 2 2 2
2 2 2 2
8.3 26.6 14.6 2 2 5.5
2 2 2 2
8.8 2.6
Description of Parafilimonas gen. nov. Parafilimonas (Pa.ra.fi.li.mo9nas. Gr. prep. para beside; N.L. fem. n. Filimonas the name of a bacterial genus; N.L. fem. n. Parafilimonas resembling the genus Filimonas). Gram-stain-negative, aerobic, non-flagellated, catalase-positive and oxidase-negative. Cells are short rods. The only menaquinone is MK-7. The polar lipids comprise a large amount of PE, and moderate and small amounts of five unknown aminolipids and seven unknown lipids. The predominant cellular fatty acids are iso-C15 : 0, iso-C17 : 0 3-OH and iso-C15 : 1 G. A member of the family Chitinophagaceae. The type species is Parafilimonas terrae. Description of Parafilimonas terrae sp. nov. Parafilimonas terrae (ter9rae. L. gen. n. terrae of/from soil). http://ijs.sgmjournals.org
*Summed features represent groups of two or more fatty acids that could not be separated by GC with the MIDI system. Summed feature 3 consisted of one or more of C16 : 1v7c, C16 : 1v6c and iso-C15 : 0 2-OH; summed feature 4 consisted of anteiso-C17 : 1 B and/or iso-C17 : 1 I.
Displays the following properties in addition to those given in the genus description. Cells are approximately 0.65– 0.75 mm61.2–2.0 mm. Colonies are irregular and yellow on R2A. Growth occurs at 15–40 uC (optimum 28–30 uC) and pH 5.0–8.0 (optimum, pH 7.0). Grows in the presence of 0–1 % (w/v) NaCl (optimum 1 %). Hydrolyses casein, DNA, Tween 80 and tyrosine, but not CM-cellulose, hypoxanthine, starch or xanthine. Produces flexirubin pigment. Positive reactions for aesculin hydrolysis and gelatin hydrolysis, but negative results for nitrate reduction, indole 3043
S.-J. Kim and others
production, glucose fermentation, arginine dihydrolase and urease (API 20NE test strips). Assimilate D-glucose, Larabinose, D-mannose, N-acetylglucosamine, maltose, sucrose, glycogen, salicin and melibiose, but not D-mannitol, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate, phenylacetic acid, L-rhamnose, D-ribose, inositol, itaconic acid, suberic acid, sodium malonate, sodium acetate, lactic acid, L-alanine, potassium 5-ketogluconate, 3-hydroxybenzoic acid, L-serine, L-fucose, D-sorbitol, propionic acid, valeric acid, L-histidine, potassium 2ketogluconate, 3-hydroxybutyric acid, 4-hydroxybenzoic acid or L-proline (API 20NE and API ID 32GN test strips). Positive results for activities of alkaline phosphatase, leucine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, a-galactosidase, b-galactosidase, N-acetyl-b-glucosaminidase and a-fucosidase, but negative results for esterase (C4), esterase lipase (C8), lipase (C14), valine arylamidase, cystine arylamidase, trypsin, a-chymotrypsin, b-glucuronidase, a-glucosidase, b-glucosidase and a-mannosidase activities (API ZYM test strips). The type strain is 5GHs7-2T (5KACC 17343T5DSM 28286T), isolated from greenhouse soil at Yongjin–myeon, Wanju-gun, Republic of Korea. The DNA G+C content of the type strain is 47.6 mol%.
Acknowledgements This study was carried out with the support of the National Academy of Agricultural Science, Rural Development Administration, Republic of Korea (project no. PJ008666).
Jin, D., Wang, P., Bai, Z., Jin, B., Yu, Z., Wang, X., Zhuang, G. & Zhang, H. (2013). Terrimonas pekingensis sp. nov., isolated from bulking
sludge, and emended descriptions of the genus Terrimonas, Terrimonas ferruginea, Terrimonas lutea and Terrimonas aquatica. Int J Syst Evol Microbiol 63, 1658–1664. Ka¨mpfer, P., Young, C. C., Sridhar, K. R., Arun, A. B., Lai, W. A., Shen, F. T. & Rekha, P. D. (2006). Transfer of [Flexibacter] sancti,
[Flexibacter] filiformis, [Flexibacter] japonensis and [Cytophaga] arvensicola to the genus Chitinophaga and description of Chitinophaga skermanii sp. nov. Int J Syst Evol Microbiol 56, 2223–2228.
Ka¨mpfer, P., Lodders, N. & Falsen, E. (2011). Hydrotalea flava gen. nov., sp. nov., a new member of the phylum Bacteroidetes and allocation of the genera Chitinophaga, Sediminibacterium, Lacibacter, Flavihumibacter, Flavisolibacter, Niabella, Niastella, Segetibacter, Parasegetibacter, Terrimonas, Ferruginibacter, Filimonas and Hydrotalea to the family Chitinophagaceae fam. nov. Int J Syst Evol Microbiol 61, 518–523. Kim, O. S., Cho, Y. J., Lee, K., Yoon, S. H., Kim, M., Na, H., Park, S. C., Jeon, Y. S., Lee, J. H. & other authors (2012). Introducing EzTaxon-e:
a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol 62, 716– 721. Kluge, A. G. & Farris, J. S. (1969). Quantitative phyletics and the
evolution of anurans. Syst Zool 18, 1–32. Leandro, T., Franc¸a, L., Nobre, M. F., Rainey, F. A. & da Costa, M. S. (2013). Heliimonas saccharivorans gen. nov., sp. nov., a member of the
family Chitinophagaceae isolated from a mineral water aquifer, and emended description of Filimonas lacunae. Int J Syst Evol Microbiol 63, 3793–3799. Ludwig, W., Strunk, O., Westram, R., Richter, L., Meier, H., Yadhukumar, Buchner, A., Lai, T., Steppi, S. & other authors (2004). ARB: a software environment for sequence data. Nucleic Acids
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