International Journal of Systematic and Evolutionary Microbiology (2014), 64, 1359–1364
DOI 10.1099/ijs.0.058594-0
Crenotalea thermophila gen. nov., sp. nov., a member of the family Chitinophagaceae isolated from a hot spring Satoshi Hanada, Hideyuki Tamaki, Kazunori Nakamura and Yoichi Kamagata Correspondence Satoshi Hanada
Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan
[email protected]
A thermophilic aerobic bacterium designated strain STH-1-Y1T was isolated from sulfur-turf in a Japanese hot spring (Okuhodaka hot spring, Gifu Pref.). Colonies of strain STH-1-Y1T were yellow and low convex morphology with a slightly irregular fringe. Cells were slender long rods, 0.4–0.6 mm wide and 1.2–3.0 mm long. The isolate was an obligate aerophilic organism, and could not grow by fermentation or nitrate respiration. The isolate had a thermophilic trait, and could grow at 35–60 6C and pH 5.5–7.5; maximum growth occurred at 55 6C and pH 7.0 with a doubling time of 1.9 h. The Biolog and API tests suggested that strain STH-1-Y1T was able to use various sugars such as glucose, lactose, mannose, maltose, trehalose, cellobiose and sucrose, but could not use sugar alcohols other than glycerol, i.e. adonitol, arabitol, erythritol, inositol, mannitol, sorbitol and xylitol. Lactate and glutamate could be used, but other fatty acids, i.e. acetate, citrate, propionate and succinate could not. Gelatin, casein, starch and glycogen were hydrolysed, but neither chitin nor agar was degraded. Cells lacked flexirubin and showed oxidase and catalase activities. The major respiratory quinone was menaquinone-7 (MK-7), and major cellular fatty acids were iso-C15 : 0, iso-C17 : 0 3-OH, iso-C17 : 0 and anteiso-C15 : 0. No unsaturated fatty acids were detected. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain STH-1-Y1T was closely related to the family Chitinophagaceae within the phylum Bacteroidetes. However, the isolate was evenly distant from all members in this family with sequence similarities of 87–89 %. These significantly low sequence similarities strongly suggested that strain STH-1-Y1T represents a novel species in a new genus of the family Chitinophagaceae within the phylum Bacteroidetes. Based on phenotypic and phylogenetic characteristics, the name Crenotalea thermophila gen. nov., sp. nov. is proposed. The type strain of the type species is STH-1-Y1T (5JCM 11541T5DSM 14807T).
The family Chitinophagaceae in the phylum Bacteroidetes was recently established by Ka¨mpfer et al. (2011) with Chitinophaga as the type genus. This family consisted of thirteen genera at this proposal: Chitinophaga (Ka¨mpfer et al., 2006; Sangkhobol & Skerman, 1981), Sediminibacterium (Qu & Yuan, 2008), Lacibacter (Qu et al., 2009), Flavihumibacter (Zhang et al., 2010b), Flavisolibacter (Yoon & Im, 2007), Niabella (Dai et al., 2011; Kim et al., 2007), Niastella (Weon et al., 2006; Zhang et al., 2010a), Segetibacter (An et al., 2007), Parasegetibacter (Zhang et al., 2009), Terrimonas (Xie & Yokota, 2006), Ferruginibacter (Lim et al., 2009), Filimonas (Shiratori et al., 2009) and Hydrotalea (Ka¨mpfer et al., 2011). On the heels of the establishment of this family, four additional novel genera were added: Flavitalea (Wang et al., 2011), Asinibacterium (Lee et al., 2013), Heliimonas (Leandro The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain STH-1-Y1T is AB849120.
058594 G 2014 IUMS
Printed in Great Britain
et al., 2013) and Taibaiella (Zhang et al., 2013). Accordingly, seventeen genera are recognized to be constituents of the family Chitinophagaceae at the time of writing. Two other genera, Balneola and Gracilimonas, are also classified in this family according to the List of Prokaryotic Names with Standing in Nomenclature (LPSN; http://www.bacterio.net/ ), but they are obviously distant from all other members of the family Chitinophagaceae in respect of the phylogenetic analysis based on 16S rRNA gene sequences. The family Chitinophagaceae possesses not only a large number of genera but also wide phylogenetic extent (the minimum 16S rRNA gene sequence similarity between species in the family was approx. 90 %). In spite of the phylogenetic diversity, all members of the family Chitinophagaceae wholly share the common feature of being mesophilic; the upper limit of growth temperature of the family is 37–45 uC, and the majority of members cannot even tolerate this temperature. Strain STH-1-Y1T showing significant growth at up to 60 uC 1359
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was isolated from a Japanese hot spring, and 16S rRNA gene sequence analysis suggested that the strain was closely related to the family Chitinophagaceae within the phylum Bacteroidetes. Strain STH-1-Y1T is, thus, considered the first discovered thermophile in the neighbourhood of the family Chitinophagaceae that has until now been occupied by mesophilic members, and warrants the creation of a novel species in a new genus of the family Chitinophagaceae. The origin of strain STH-1-Y1T was sulfur-turf, a microbial mat mainly formed by a species of the genus Sulfurihydrogenibium and elemental sulfur particles, in the stream of a hot spring (Okuhodaka hot spring, Gifu prefecture, Japan). A piece of the sulfur-turf developed in neutral hot spring water at 65 uC was streaked on PE agar medium (Hanada et al., 1995) adjusted to pH 7.5. The inoculated plate was incubated at 55 uC under aerobic conditions. Yellow colonies of strain STH-1-Y1T emerged on the plate after 2 weeks. A liquid PE medium (pH 7.0) was generally used to grow the isolated strain. Colony morphology and gliding motility on an agar plate were observed with a stereo-microscope (SZX12; Olympus), and cell morphology was examined using a phase-contrast microscopy (AX80; Olympus). A method for transmission electron microscopy was minutely described in a previous paper (Hanada et al., 2002). Growth at 20–75 uC (in 5 uC increments) was tested using PE medium at pH 7, and growth at pH 5.0–8.0 (in 0.5 pH unit increments) was investigated at 55 uC in PE medium with appropriate buffering agents (for pH 5.0–5.5, 10 mM sodium acetate buffer; for pH 6.0–8.0, 10 mM potassium phosphate buffer). Organic substrate oxidation, enzyme activity and some biochemical characteristics were determined by API 20E, API ID32E and API ZYM kits (bioMe´rieux) as well as the Biolog system GN2 and GP2 microplates (Biolog). The presence of flexirubin was ascertained according to the method of Gu¨de (1980). Chitin hydrolysis was tested with chitin agar containing 0.4 % crab shell chitin (Wako Chemicals) in colloidal form (Smibert & Krieg, 1994). Gram-staining, oxidase activity, catalase production, nitrate reduction, fermentative growth under anaerobic conditions, respiratory quinones and DNA base composition were analysed as described previously (Shintani et al., 2000). Analysis of fatty acid methyl esters with a GC-MS was also detailed in a previous report (Hanada et al., 2002). The 16S rRNA gene sequence of strain STH-1-Y1T was determined as described previously (Shintani et al., 2000). The phylogenetic tree was calculated based on a distance matrix analysis of 16S rRNA gene sequences obtained from the SILVA database (neighbour-joining tree as inferred with the ARB program package). Bootstrap resampling analysis of 1000 replicates with the neighbour-joining (PAUP*), maximum-parsimony (PAUP*) and maximum-likelihood (RAxML) methods were performed to estimate the confidence of the tree topologies.
While the isolate was related to the family, it was evenly distant from all members of this family with 16S rRNA gene sequence similarities of 87–89 %. The high bootstrap values (100 % neighbour-joining, 100 % maximum-likelihood and 100 % maximum-parsimony) of the branching point between the genus Taibaiella and all other members of the family Chitinophagaceae also supported the classification of strain STH-1-Y1T in this family. The significantly low sequence similarities strongly suggested that strain STH-1Y1T represents a novel species of a new genus in the family Chitinophagaceae within the phylum Bacteroidetes. Colonies of strain STH-1-Y1T showed a low convex morphology with a slight irregular fringe; a yellow pigment was generally produced. Cells of strain STH-1-Y1T were slender long rods, 0.4–0.6 mm wide and 1.2–3.0 mm long (Fig. 2a). Electron microscopy of a thin section revealed that cells had neither intracellular vesicles nor invagination of inner membrane (Fig. 2b). The isolate was an obligate aerophilic organism unable to grow by fermentation or nitrate respiration. Growth occurred at 35–60 uC and pH 5.5–7.5; maximum growth was observed at 55 uC and pH 7.0 with a doubling time of 1.9 h. Strain STH-1-Y1T could not grow at 30 and 65 uC or pH 5 and 8. Biolog and API tests suggested that strain STH-1-Y1T had a unique inclination in nutrient utilization: it was able to use various sugars such as glucose, lactose, mannose, maltose, trehalose, cellobiose and sucrose, but sugar alcohols other than glycerol (adonitol, arabitol, erythritol, inositol, mannitol, sorbitol and xylitol) were not used; although lactate and glutamate could be used, other fatty acids such as acetate, citrate, propionate and succinate could not. The isolate could hydrolyse gelatin, casein, starch and glycogen. Neither chitin degradation nor agar liquefaction was observed. APIZYM tests revealed activity of the following enzymes: alkaline phosphatase, leucine arylamidase, acidic phosphatase, naphthol-AS-BI-phosphohydrolase, b-galactosidase, aglucosidase and N-acetyl-b-glucosaminidase. Other physiological properties such as the absence of flexirubin and the presence of oxidase and catalase activities are summarized in Table 1.
Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain STH-1-Y1T was placed in the family Chitinophagaceae within the phylum Bacteroidetes (Fig. 1).
The major respiratory quinone of strain STH-1-Y1T was menaquinone-7 (MK-7) which is typical of all members of the family Chitinophagaceae (Table 1). Analysis of fatty acid methyl esters with a GC-MS revealed that the cellular fatty acid composition of strain STH-1-Y1T was occupied by branched fatty acids with odd number of carbons, i.e. isobranched C15 : 0 (34 %) and C17 : 0 (17 %), and anteisobranched C15 : 0 (13 %) and C17 : 0 (3 %). This tendency was commonly found in members of the family Chitinophagaceae (Table 1). Also, iso-C17 : 0 3-OH, the typical fatty acid of this family, was contained as the second dominant component (22 %). However, strain STH-1-Y1T did not contain iso-C15 : 1, an unsaturated fatty acid generally detected in members of the family Chitinophagaceae. The absence of unsaturated fatty acids, which would be connected with its thermophily, is a remarkable feature to differentiate the isolate from members of the family Chitinophagaceae.
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International Journal of Systematic and Evolutionary Microbiology 64
Crenotalea thermophila gen. nov., sp. nov.
0.05
Asinibacterium lactis LCJ02T (JQ638910) Sediminibacterium salmoneum NJ-44T (EF407879) Hydrotalea flava CCUG 51397T (FN665659) Flavihumibacter petaseus T41T (EU854577) Parasegetibacter luojiensis RHYL-37T (EU877263) Filimonas lacunae YT21T (AB362776) Segetibacter koreensis Gsoil 664T (AB267478) Flavisolibacter ginsengiterrae Gsoil 492T (AB267476) Flavitalea populi HY-50RT (HM130561) Niabella aurantiaca R2A15-11T (DQ457019) Terrimonas ferruginea DSM 30193T (AM230484) Niastella koreensis GR20-10T (DQ244077) Heliimonas saccharivorans L2-4T (JX458466) Lacibacter cauensis NJ-8T (EU521690) Ferruginibacter alkalilentus HU1-GD23T (FJ177530) Chitinophaga pinensis DSM 2588T (AF078775) Crenotalea thermophila STH-1-Y1T (AB849120) Taibaiella smilacinae PTJT-5T (KC571459) Haliscomenobacter hydrossis DSM 1100T (AJ784892) Lewinella cohaerens ATCC 23123T (AF039292) Aureispira marina 24T (AB245933) Cryomorpha ignava ACAM 647T (AF170738) Owenweeksia hongkongensis UST20020801T (AB125062) Balneola vulgaris 13IX/A01/164T (AY576749) Gracilimonas tropica CL-CB462T (EF988655)
Chitinophagaceae
Saprospiraceae Cryomorphaceae Chitinophagaceae
Fig. 1. Phylogenetic relationship between strain STH-1-Y1T and the family Chitinophagaceae within the phylum Bacteroidetes. The tree was calculated based on a distance matrix analysis of 16S rRNA gene sequences obtained from the SILVA database (neighbour-joining tree as inferred with the ARB program package). Bootstrap resampling analyses of 1000 replicates with neighbour-joining (PAUP*), maximum-parsimony (PAUP*) and maximum-likelihood (RAxML) methods were performed to estimate the confidence of the tree topologies. Branching points with supported probabilities .90 % by all the analyses are indicated by a filled circle, whereas nodes with open circles indicate .70 % bootstrap probabilities supported by all the analyses, respectively. Bar, 0.05 nucleotide changes per sequence position.
Based on phylogenetic and phenotypic comparisons, a new taxon within the family Chitinophagaceae in the phylum Bacteroidetes is created for strain STH-1-Y1T with the name, Crenotalea thermophila gen. nov., sp. nov. The novel species is phylogenetically related to the family Chitinophagaceae and shares several common features with members of this family, e.g. yellowish pigmentation, MK7 as major respiratory quinone, branched fatty acids with odd number of carbons occupying the cellular fatty acids and the presence of a distinctive methoxy-fatty acid (isoC17 : 0 3-OH). However, not only a significant distance of
(a)
(b)
16S rRNA gene sequence but also the following obvious phenotypic characteristics differentiate the novel genus from recognized genera of the family Chitinophagaceae: the organism is a thermophile, able to grow at up to 60 uC, and lacks an unsaturated fatty acid (iso-C15 : 1) commonly found in members of the family. Some related strains which have not yet been taxonomically classified are hit in the BLAST search with significantly high sequence similarities (approx. 99 %). These closely related strains, Bacteroidetes bacterium P373 (GenBank accession no. AM749771) and Bacteroidetes bacterium K3 (AM749788) have been isolated from geothermal soil in New Zealand (Stott et al., 2008) and are probably thermophilic like strain STH-1-Y1T. Whereas their phenotypic characteristics have not been reported at all, these New Zealand strains would most likely be classified into the same genus (or the same species) as STH-1-Y1T. Description of Crenotalea gen. nov. Crenotalea (Cre.no.ta9le.a. Gr. n. krene a spring; L. fem. n. talea a slender staff, a rod; N.L. fem. n. Crenotalea a slender rod in a hot spring).
Fig. 2. Phase-contrast photomicrograph (a) and electron micrograph of ultrathin section (b) of cells of strain STH-1-Y1T grown in PE medium (pH 7.0) at 55 6C under aerobic conditions for 3 days. Bars, 10 mm (a) and 0.5 mm (b). http://ijs.sgmjournals.org
Gram-stain-negative, non-motile, aerobic bacterium with slender rod-shaped cell morphology. Yellow pigmentation. Thermophile, able to grow at up to 60 uC. Major fatty acids are iso-C15 : 0, iso-C17 : 0 3-OH and iso-C17 : 0. No 1361
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Table 1. Differential characteristics of strain STH-1-Y1T and seventeen genera of the family Chitinophagaceae Genera: 1, Crenotalea gen. nov. (strain STH-1-Y1T; data from this study); 2, Sediminibacterium (Qu & Yuan, 2008); 3, Flavisolibacter (Yoon & Im, 2007); 4, Lacibacter (Qu et al., 2009); 5, Flavihumibacter (Zhang et al., 2010b, 2013); 6, Terrimonas (Xie & Yokota, 2006); 7, Niabella (Dai et al., 2011); 8, Niastella (Weon et al., 2006; Zhang et al., 2010a); 9, Segetibacter (An et al., 2007); 10, Chitinophaga (Chung et al., 2012; Ka¨mpfer et al., 2006; Sangkhobol & Skerman, 1981); 11, Filimonas (Shiratori et al., 2009); 12, Parasegetibacter (Zhang et al., 2009); 13, Ferruginibacter (Lim et al., 2009); 14, Hydrotalea (Ka¨mpfer et al., 2011); 15, Flavitalea (Wang et al., 2011); 16, Asinibacterium (Lee et al., 2013); 17, Heliimonas (Leandro et al., 2013); 18, Taibaiella (Zhang et al., 2013). +, All species positive; –, all species negative; W, weakly positive; V, variable among species; ND, no data available.
International Journal of Systematic and Evolutionary Microbiology 64
Characteristic
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Growth temperature (uC) Range Optimum
35–60 55
18–37 22–28
15-37 30
20–37 28–37
20–37 28
10–37 25–32
10–35 25–30
10–30
15–30
10–35 28–30
18–30
20–37
ND
15–40 mostly 25–30
17–37
ND
ND
ND
ND
15–37 30
20–30 25
10–45 30
7–33 25–28
– + +
– + +
ND
– + +
+
–
ND
V
– + +
– + +
– + +
– + +
– – +
– – –
– + +
+ + +
– – – – MK-7 i-C15 : 0, i-C17 : 0 3-OH, i-C15 : 1
– + + – MK-7 i-C17 : 0 3-OH, i-C15 : 0, i-C15 : 1
– + + + MK-7 i-C15 : 0, i-C15 : 1, i-C17 : 0 3-OH
49.2
42.0
40.3
Flexirubin Catalase Oxidase Hydrolysis of: Chitin Casein Starch Gelatin Quinone system Major cellular fatty acids
Genomic DNA G+C content (mol%)
V V
– – – + – – + – – + + + ND MK-7 MK-7 i-C15 : 0, i-C15 : 0, i-C15 : 0, ii-C17 : 0 i-C15 : 1, C17 : 0 33-OH, i-C17 : 0 OH, ii-C17 : 0 3-OH C15 : 1 46.6
40.6
42.7–43.0
– + + + MK-7 i-C15 : 0, i-C15 : 1, i-C17 : 0 3-OH 46.6
W
+
V
+ W
– – + V V – + V MK-7 MK-7 i-C15 : 0, i-C15 : 0, ii-C15 : 1, C15 : 1, ii-C17 : 0 C17 : 0 3OH 3-OH 48.1
47.2–48.9
+ – + –
V
–
– – – – MK-7 i-C15 : 0, i-C17 : 0 3-OH, i-C15 : 1, i-C16 : 1 44.3–45.8 40.4 V
+ V – V + MK-7 MK-7 i-C15 : 0, i-C15 : 0, ii-C15 : 1, C15 : 1, ii-C17 : 0 C17 : 0 3OH 3-OH V
45.0
+ +
V V
– – V – V + MK-7 MK-7 i-C15 : 0, i- i-C15 : 0, C16 : 1, i- i-C17 : 0 C17 : 0 3- 3-OH, OH i-C15 : 1 V V
40.7–49.8
45.2
– – – – + – + + MK-7 MK-7 i-C15 : 0, i-C15 : 0, ii-C17 : 0 C17 : 0 33-OH, OH, ii-C15 : 1, C15 : 1, iC16 : 0 C17 : 1 39.7 38.5–39.5
– – ND – ND – MK-7 MK-7 i-C15 : 0, i-C15 : 0, i-C17 : 0 i-C17 : 0 3-OH, 3-OH, i-C15 : 1 i-C15 : 1 ND ND
42.0
46.8
Crenotalea thermophila gen. nov., sp. nov.
unsaturated fatty acids are detected. Major respiratory quinone is menaquinone-7. The type species is Crenotalea thermophile. The genomic DNA G+C content of the type species is 46.6 mol%.
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.,
Description of Crenotalea thermophila sp. nov. Crenotalea thermophila (ther.mo9phi.la. Gr. adj. thermos hot; N.L. adj. philus -a -um (from Gr. adj. philos -eˆ -on) friend, loving; N.L. fem. adj. thermophila heat-loving). Gram-stain-negative, non-motile, slender rod-shaped cells, 0.4–0.6 mm wide and 1.2–3.0 mm long. Obligate aerophilic organism unable to grow by fermentation or nitrate respiration. Thermophile. Growth occurs at 35–60 uC and pH 5.5– 7.5; maximum growth is observed at 55 uC and pH 7.0 with a doubling time of 1.9 h. Utilizes various sugars such as glucose, lactose, mannose, maltose, trehalose, cellobiose and sucrose, but sugar alcohols other than glycerol (adonitol, arabitol, erythritol, inositol, mannitol, sorbitol and xylitol) cannot be used. Lactate and glutamate can be used, but acetate, citrate, propionate and succinate cannot be used. Gelatin, casein, starch and glycogen are hydrolysed. Neither chitin nor agar is degraded. Flexirubin is absent. Oxidase and catalase are produced. Voges–Proskauer test is positive. Tests for indole production, H2S production and urease activity are negative. Menaquinone-7 is the predominant respiratory quinone. Cellular fatty acids are iso-C15 : 0, isoC17 : 0 3-OH, iso-C17 : 0, anteiso-C15 : 0, iso-C16 : 0, C14 : 0 and anteiso-C17 : 0. The type strain is strain STH-1-Y1T (5JCM 11541T5DSM 14807T), isolated from sulfur-turf, a microbial mat mainly formed by a species of the genus Sulfurihydrogenibium and elemental sulfur particles, developed at 65 uC in a Japanese hot spring (Okuhodaka, Gifu prefecture). The genomic DNA G+C content of the type strain is 46.6 mol%.
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from soil of Euphrates poplar (Populus euphratica) forest, and emended description of the genus Niastella. Int J Syst Evol Microbiol 60, 542–545. Zhang, N. N., Qu, J. H., Yuan, H. L., Sun, Y. M. & Yang, J. S. (2010b).
Zhang, L., Wang, Y., Wei, L., Wang, Y., Shen, X. & Li, S. (2013).
member of the phylum Bacteroidetes isolated from a forest soil. Int J Syst Evol Microbiol 59, 3058–3062.
Taibaiella smilacinae gen. nov., sp. nov., an endophytic member of the family Chitinophagaceae isolated from the stem of Smilacina japonica and emended description of Flavihumibacter petaseus. Int J Syst Evol Microbiol 63, 3769–3776.
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Zhang, K., Tang, Y., Zhang, L., Dai, J., Wang, Y., Luo, X., Liu, M., Luo, G. & Fang, C. (2009). Parasegetibacter luojiensis gen. nov., sp. nov., a
DOI 10.1099/ijs.0.058594-0
Crenotalea thermophila gen. nov., sp. nov., a member of the family Chitinophagaceae isolated from a hot spring Satoshi Hanada, Hideyuki Tamaki, Kazunori Nakamura and Yoichi Kamagata Correspondence Satoshi Hanada
Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan
[email protected]
A thermophilic aerobic bacterium designated strain STH-1-Y1T was isolated from sulfur-turf in a Japanese hot spring (Okuhodaka hot spring, Gifu Pref.). Colonies of strain STH-1-Y1T were yellow and low convex morphology with a slightly irregular fringe. Cells were slender long rods, 0.4–0.6 mm wide and 1.2–3.0 mm long. The isolate was an obligate aerophilic organism, and could not grow by fermentation or nitrate respiration. The isolate had a thermophilic trait, and could grow at 35–60 6C and pH 5.5–7.5; maximum growth occurred at 55 6C and pH 7.0 with a doubling time of 1.9 h. The Biolog and API tests suggested that strain STH-1-Y1T was able to use various sugars such as glucose, lactose, mannose, maltose, trehalose, cellobiose and sucrose, but could not use sugar alcohols other than glycerol, i.e. adonitol, arabitol, erythritol, inositol, mannitol, sorbitol and xylitol. Lactate and glutamate could be used, but other fatty acids, i.e. acetate, citrate, propionate and succinate could not. Gelatin, casein, starch and glycogen were hydrolysed, but neither chitin nor agar was degraded. Cells lacked flexirubin and showed oxidase and catalase activities. The major respiratory quinone was menaquinone-7 (MK-7), and major cellular fatty acids were iso-C15 : 0, iso-C17 : 0 3-OH, iso-C17 : 0 and anteiso-C15 : 0. No unsaturated fatty acids were detected. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain STH-1-Y1T was closely related to the family Chitinophagaceae within the phylum Bacteroidetes. However, the isolate was evenly distant from all members in this family with sequence similarities of 87–89 %. These significantly low sequence similarities strongly suggested that strain STH-1-Y1T represents a novel species in a new genus of the family Chitinophagaceae within the phylum Bacteroidetes. Based on phenotypic and phylogenetic characteristics, the name Crenotalea thermophila gen. nov., sp. nov. is proposed. The type strain of the type species is STH-1-Y1T (5JCM 11541T5DSM 14807T).
The family Chitinophagaceae in the phylum Bacteroidetes was recently established by Ka¨mpfer et al. (2011) with Chitinophaga as the type genus. This family consisted of thirteen genera at this proposal: Chitinophaga (Ka¨mpfer et al., 2006; Sangkhobol & Skerman, 1981), Sediminibacterium (Qu & Yuan, 2008), Lacibacter (Qu et al., 2009), Flavihumibacter (Zhang et al., 2010b), Flavisolibacter (Yoon & Im, 2007), Niabella (Dai et al., 2011; Kim et al., 2007), Niastella (Weon et al., 2006; Zhang et al., 2010a), Segetibacter (An et al., 2007), Parasegetibacter (Zhang et al., 2009), Terrimonas (Xie & Yokota, 2006), Ferruginibacter (Lim et al., 2009), Filimonas (Shiratori et al., 2009) and Hydrotalea (Ka¨mpfer et al., 2011). On the heels of the establishment of this family, four additional novel genera were added: Flavitalea (Wang et al., 2011), Asinibacterium (Lee et al., 2013), Heliimonas (Leandro The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain STH-1-Y1T is AB849120.
058594 G 2014 IUMS
Printed in Great Britain
et al., 2013) and Taibaiella (Zhang et al., 2013). Accordingly, seventeen genera are recognized to be constituents of the family Chitinophagaceae at the time of writing. Two other genera, Balneola and Gracilimonas, are also classified in this family according to the List of Prokaryotic Names with Standing in Nomenclature (LPSN; http://www.bacterio.net/ ), but they are obviously distant from all other members of the family Chitinophagaceae in respect of the phylogenetic analysis based on 16S rRNA gene sequences. The family Chitinophagaceae possesses not only a large number of genera but also wide phylogenetic extent (the minimum 16S rRNA gene sequence similarity between species in the family was approx. 90 %). In spite of the phylogenetic diversity, all members of the family Chitinophagaceae wholly share the common feature of being mesophilic; the upper limit of growth temperature of the family is 37–45 uC, and the majority of members cannot even tolerate this temperature. Strain STH-1-Y1T showing significant growth at up to 60 uC 1359
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was isolated from a Japanese hot spring, and 16S rRNA gene sequence analysis suggested that the strain was closely related to the family Chitinophagaceae within the phylum Bacteroidetes. Strain STH-1-Y1T is, thus, considered the first discovered thermophile in the neighbourhood of the family Chitinophagaceae that has until now been occupied by mesophilic members, and warrants the creation of a novel species in a new genus of the family Chitinophagaceae. The origin of strain STH-1-Y1T was sulfur-turf, a microbial mat mainly formed by a species of the genus Sulfurihydrogenibium and elemental sulfur particles, in the stream of a hot spring (Okuhodaka hot spring, Gifu prefecture, Japan). A piece of the sulfur-turf developed in neutral hot spring water at 65 uC was streaked on PE agar medium (Hanada et al., 1995) adjusted to pH 7.5. The inoculated plate was incubated at 55 uC under aerobic conditions. Yellow colonies of strain STH-1-Y1T emerged on the plate after 2 weeks. A liquid PE medium (pH 7.0) was generally used to grow the isolated strain. Colony morphology and gliding motility on an agar plate were observed with a stereo-microscope (SZX12; Olympus), and cell morphology was examined using a phase-contrast microscopy (AX80; Olympus). A method for transmission electron microscopy was minutely described in a previous paper (Hanada et al., 2002). Growth at 20–75 uC (in 5 uC increments) was tested using PE medium at pH 7, and growth at pH 5.0–8.0 (in 0.5 pH unit increments) was investigated at 55 uC in PE medium with appropriate buffering agents (for pH 5.0–5.5, 10 mM sodium acetate buffer; for pH 6.0–8.0, 10 mM potassium phosphate buffer). Organic substrate oxidation, enzyme activity and some biochemical characteristics were determined by API 20E, API ID32E and API ZYM kits (bioMe´rieux) as well as the Biolog system GN2 and GP2 microplates (Biolog). The presence of flexirubin was ascertained according to the method of Gu¨de (1980). Chitin hydrolysis was tested with chitin agar containing 0.4 % crab shell chitin (Wako Chemicals) in colloidal form (Smibert & Krieg, 1994). Gram-staining, oxidase activity, catalase production, nitrate reduction, fermentative growth under anaerobic conditions, respiratory quinones and DNA base composition were analysed as described previously (Shintani et al., 2000). Analysis of fatty acid methyl esters with a GC-MS was also detailed in a previous report (Hanada et al., 2002). The 16S rRNA gene sequence of strain STH-1-Y1T was determined as described previously (Shintani et al., 2000). The phylogenetic tree was calculated based on a distance matrix analysis of 16S rRNA gene sequences obtained from the SILVA database (neighbour-joining tree as inferred with the ARB program package). Bootstrap resampling analysis of 1000 replicates with the neighbour-joining (PAUP*), maximum-parsimony (PAUP*) and maximum-likelihood (RAxML) methods were performed to estimate the confidence of the tree topologies.
While the isolate was related to the family, it was evenly distant from all members of this family with 16S rRNA gene sequence similarities of 87–89 %. The high bootstrap values (100 % neighbour-joining, 100 % maximum-likelihood and 100 % maximum-parsimony) of the branching point between the genus Taibaiella and all other members of the family Chitinophagaceae also supported the classification of strain STH-1-Y1T in this family. The significantly low sequence similarities strongly suggested that strain STH-1Y1T represents a novel species of a new genus in the family Chitinophagaceae within the phylum Bacteroidetes. Colonies of strain STH-1-Y1T showed a low convex morphology with a slight irregular fringe; a yellow pigment was generally produced. Cells of strain STH-1-Y1T were slender long rods, 0.4–0.6 mm wide and 1.2–3.0 mm long (Fig. 2a). Electron microscopy of a thin section revealed that cells had neither intracellular vesicles nor invagination of inner membrane (Fig. 2b). The isolate was an obligate aerophilic organism unable to grow by fermentation or nitrate respiration. Growth occurred at 35–60 uC and pH 5.5–7.5; maximum growth was observed at 55 uC and pH 7.0 with a doubling time of 1.9 h. Strain STH-1-Y1T could not grow at 30 and 65 uC or pH 5 and 8. Biolog and API tests suggested that strain STH-1-Y1T had a unique inclination in nutrient utilization: it was able to use various sugars such as glucose, lactose, mannose, maltose, trehalose, cellobiose and sucrose, but sugar alcohols other than glycerol (adonitol, arabitol, erythritol, inositol, mannitol, sorbitol and xylitol) were not used; although lactate and glutamate could be used, other fatty acids such as acetate, citrate, propionate and succinate could not. The isolate could hydrolyse gelatin, casein, starch and glycogen. Neither chitin degradation nor agar liquefaction was observed. APIZYM tests revealed activity of the following enzymes: alkaline phosphatase, leucine arylamidase, acidic phosphatase, naphthol-AS-BI-phosphohydrolase, b-galactosidase, aglucosidase and N-acetyl-b-glucosaminidase. Other physiological properties such as the absence of flexirubin and the presence of oxidase and catalase activities are summarized in Table 1.
Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain STH-1-Y1T was placed in the family Chitinophagaceae within the phylum Bacteroidetes (Fig. 1).
The major respiratory quinone of strain STH-1-Y1T was menaquinone-7 (MK-7) which is typical of all members of the family Chitinophagaceae (Table 1). Analysis of fatty acid methyl esters with a GC-MS revealed that the cellular fatty acid composition of strain STH-1-Y1T was occupied by branched fatty acids with odd number of carbons, i.e. isobranched C15 : 0 (34 %) and C17 : 0 (17 %), and anteisobranched C15 : 0 (13 %) and C17 : 0 (3 %). This tendency was commonly found in members of the family Chitinophagaceae (Table 1). Also, iso-C17 : 0 3-OH, the typical fatty acid of this family, was contained as the second dominant component (22 %). However, strain STH-1-Y1T did not contain iso-C15 : 1, an unsaturated fatty acid generally detected in members of the family Chitinophagaceae. The absence of unsaturated fatty acids, which would be connected with its thermophily, is a remarkable feature to differentiate the isolate from members of the family Chitinophagaceae.
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Crenotalea thermophila gen. nov., sp. nov.
0.05
Asinibacterium lactis LCJ02T (JQ638910) Sediminibacterium salmoneum NJ-44T (EF407879) Hydrotalea flava CCUG 51397T (FN665659) Flavihumibacter petaseus T41T (EU854577) Parasegetibacter luojiensis RHYL-37T (EU877263) Filimonas lacunae YT21T (AB362776) Segetibacter koreensis Gsoil 664T (AB267478) Flavisolibacter ginsengiterrae Gsoil 492T (AB267476) Flavitalea populi HY-50RT (HM130561) Niabella aurantiaca R2A15-11T (DQ457019) Terrimonas ferruginea DSM 30193T (AM230484) Niastella koreensis GR20-10T (DQ244077) Heliimonas saccharivorans L2-4T (JX458466) Lacibacter cauensis NJ-8T (EU521690) Ferruginibacter alkalilentus HU1-GD23T (FJ177530) Chitinophaga pinensis DSM 2588T (AF078775) Crenotalea thermophila STH-1-Y1T (AB849120) Taibaiella smilacinae PTJT-5T (KC571459) Haliscomenobacter hydrossis DSM 1100T (AJ784892) Lewinella cohaerens ATCC 23123T (AF039292) Aureispira marina 24T (AB245933) Cryomorpha ignava ACAM 647T (AF170738) Owenweeksia hongkongensis UST20020801T (AB125062) Balneola vulgaris 13IX/A01/164T (AY576749) Gracilimonas tropica CL-CB462T (EF988655)
Chitinophagaceae
Saprospiraceae Cryomorphaceae Chitinophagaceae
Fig. 1. Phylogenetic relationship between strain STH-1-Y1T and the family Chitinophagaceae within the phylum Bacteroidetes. The tree was calculated based on a distance matrix analysis of 16S rRNA gene sequences obtained from the SILVA database (neighbour-joining tree as inferred with the ARB program package). Bootstrap resampling analyses of 1000 replicates with neighbour-joining (PAUP*), maximum-parsimony (PAUP*) and maximum-likelihood (RAxML) methods were performed to estimate the confidence of the tree topologies. Branching points with supported probabilities .90 % by all the analyses are indicated by a filled circle, whereas nodes with open circles indicate .70 % bootstrap probabilities supported by all the analyses, respectively. Bar, 0.05 nucleotide changes per sequence position.
Based on phylogenetic and phenotypic comparisons, a new taxon within the family Chitinophagaceae in the phylum Bacteroidetes is created for strain STH-1-Y1T with the name, Crenotalea thermophila gen. nov., sp. nov. The novel species is phylogenetically related to the family Chitinophagaceae and shares several common features with members of this family, e.g. yellowish pigmentation, MK7 as major respiratory quinone, branched fatty acids with odd number of carbons occupying the cellular fatty acids and the presence of a distinctive methoxy-fatty acid (isoC17 : 0 3-OH). However, not only a significant distance of
(a)
(b)
16S rRNA gene sequence but also the following obvious phenotypic characteristics differentiate the novel genus from recognized genera of the family Chitinophagaceae: the organism is a thermophile, able to grow at up to 60 uC, and lacks an unsaturated fatty acid (iso-C15 : 1) commonly found in members of the family. Some related strains which have not yet been taxonomically classified are hit in the BLAST search with significantly high sequence similarities (approx. 99 %). These closely related strains, Bacteroidetes bacterium P373 (GenBank accession no. AM749771) and Bacteroidetes bacterium K3 (AM749788) have been isolated from geothermal soil in New Zealand (Stott et al., 2008) and are probably thermophilic like strain STH-1-Y1T. Whereas their phenotypic characteristics have not been reported at all, these New Zealand strains would most likely be classified into the same genus (or the same species) as STH-1-Y1T. Description of Crenotalea gen. nov. Crenotalea (Cre.no.ta9le.a. Gr. n. krene a spring; L. fem. n. talea a slender staff, a rod; N.L. fem. n. Crenotalea a slender rod in a hot spring).
Fig. 2. Phase-contrast photomicrograph (a) and electron micrograph of ultrathin section (b) of cells of strain STH-1-Y1T grown in PE medium (pH 7.0) at 55 6C under aerobic conditions for 3 days. Bars, 10 mm (a) and 0.5 mm (b). http://ijs.sgmjournals.org
Gram-stain-negative, non-motile, aerobic bacterium with slender rod-shaped cell morphology. Yellow pigmentation. Thermophile, able to grow at up to 60 uC. Major fatty acids are iso-C15 : 0, iso-C17 : 0 3-OH and iso-C17 : 0. No 1361
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Table 1. Differential characteristics of strain STH-1-Y1T and seventeen genera of the family Chitinophagaceae Genera: 1, Crenotalea gen. nov. (strain STH-1-Y1T; data from this study); 2, Sediminibacterium (Qu & Yuan, 2008); 3, Flavisolibacter (Yoon & Im, 2007); 4, Lacibacter (Qu et al., 2009); 5, Flavihumibacter (Zhang et al., 2010b, 2013); 6, Terrimonas (Xie & Yokota, 2006); 7, Niabella (Dai et al., 2011); 8, Niastella (Weon et al., 2006; Zhang et al., 2010a); 9, Segetibacter (An et al., 2007); 10, Chitinophaga (Chung et al., 2012; Ka¨mpfer et al., 2006; Sangkhobol & Skerman, 1981); 11, Filimonas (Shiratori et al., 2009); 12, Parasegetibacter (Zhang et al., 2009); 13, Ferruginibacter (Lim et al., 2009); 14, Hydrotalea (Ka¨mpfer et al., 2011); 15, Flavitalea (Wang et al., 2011); 16, Asinibacterium (Lee et al., 2013); 17, Heliimonas (Leandro et al., 2013); 18, Taibaiella (Zhang et al., 2013). +, All species positive; –, all species negative; W, weakly positive; V, variable among species; ND, no data available.
International Journal of Systematic and Evolutionary Microbiology 64
Characteristic
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Growth temperature (uC) Range Optimum
35–60 55
18–37 22–28
15-37 30
20–37 28–37
20–37 28
10–37 25–32
10–35 25–30
10–30
15–30
10–35 28–30
18–30
20–37
ND
15–40 mostly 25–30
17–37
ND
ND
ND
ND
15–37 30
20–30 25
10–45 30
7–33 25–28
– + +
– + +
ND
– + +
+
–
ND
V
– + +
– + +
– + +
– + +
– – +
– – –
– + +
+ + +
– – – – MK-7 i-C15 : 0, i-C17 : 0 3-OH, i-C15 : 1
– + + – MK-7 i-C17 : 0 3-OH, i-C15 : 0, i-C15 : 1
– + + + MK-7 i-C15 : 0, i-C15 : 1, i-C17 : 0 3-OH
49.2
42.0
40.3
Flexirubin Catalase Oxidase Hydrolysis of: Chitin Casein Starch Gelatin Quinone system Major cellular fatty acids
Genomic DNA G+C content (mol%)
V V
– – – + – – + – – + + + ND MK-7 MK-7 i-C15 : 0, i-C15 : 0, i-C15 : 0, ii-C17 : 0 i-C15 : 1, C17 : 0 33-OH, i-C17 : 0 OH, ii-C17 : 0 3-OH C15 : 1 46.6
40.6
42.7–43.0
– + + + MK-7 i-C15 : 0, i-C15 : 1, i-C17 : 0 3-OH 46.6
W
+
V
+ W
– – + V V – + V MK-7 MK-7 i-C15 : 0, i-C15 : 0, ii-C15 : 1, C15 : 1, ii-C17 : 0 C17 : 0 3OH 3-OH 48.1
47.2–48.9
+ – + –
V
–
– – – – MK-7 i-C15 : 0, i-C17 : 0 3-OH, i-C15 : 1, i-C16 : 1 44.3–45.8 40.4 V
+ V – V + MK-7 MK-7 i-C15 : 0, i-C15 : 0, ii-C15 : 1, C15 : 1, ii-C17 : 0 C17 : 0 3OH 3-OH V
45.0
+ +
V V
– – V – V + MK-7 MK-7 i-C15 : 0, i- i-C15 : 0, C16 : 1, i- i-C17 : 0 C17 : 0 3- 3-OH, OH i-C15 : 1 V V
40.7–49.8
45.2
– – – – + – + + MK-7 MK-7 i-C15 : 0, i-C15 : 0, ii-C17 : 0 C17 : 0 33-OH, OH, ii-C15 : 1, C15 : 1, iC16 : 0 C17 : 1 39.7 38.5–39.5
– – ND – ND – MK-7 MK-7 i-C15 : 0, i-C15 : 0, i-C17 : 0 i-C17 : 0 3-OH, 3-OH, i-C15 : 1 i-C15 : 1 ND ND
42.0
46.8
Crenotalea thermophila gen. nov., sp. nov.
unsaturated fatty acids are detected. Major respiratory quinone is menaquinone-7. The type species is Crenotalea thermophile. The genomic DNA G+C content of the type species is 46.6 mol%.
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.,
Description of Crenotalea thermophila sp. nov. Crenotalea thermophila (ther.mo9phi.la. Gr. adj. thermos hot; N.L. adj. philus -a -um (from Gr. adj. philos -eˆ -on) friend, loving; N.L. fem. adj. thermophila heat-loving). Gram-stain-negative, non-motile, slender rod-shaped cells, 0.4–0.6 mm wide and 1.2–3.0 mm long. Obligate aerophilic organism unable to grow by fermentation or nitrate respiration. Thermophile. Growth occurs at 35–60 uC and pH 5.5– 7.5; maximum growth is observed at 55 uC and pH 7.0 with a doubling time of 1.9 h. Utilizes various sugars such as glucose, lactose, mannose, maltose, trehalose, cellobiose and sucrose, but sugar alcohols other than glycerol (adonitol, arabitol, erythritol, inositol, mannitol, sorbitol and xylitol) cannot be used. Lactate and glutamate can be used, but acetate, citrate, propionate and succinate cannot be used. Gelatin, casein, starch and glycogen are hydrolysed. Neither chitin nor agar is degraded. Flexirubin is absent. Oxidase and catalase are produced. Voges–Proskauer test is positive. Tests for indole production, H2S production and urease activity are negative. Menaquinone-7 is the predominant respiratory quinone. Cellular fatty acids are iso-C15 : 0, isoC17 : 0 3-OH, iso-C17 : 0, anteiso-C15 : 0, iso-C16 : 0, C14 : 0 and anteiso-C17 : 0. The type strain is strain STH-1-Y1T (5JCM 11541T5DSM 14807T), isolated from sulfur-turf, a microbial mat mainly formed by a species of the genus Sulfurihydrogenibium and elemental sulfur particles, developed at 65 uC in a Japanese hot spring (Okuhodaka, Gifu prefecture). The genomic DNA G+C content of the type strain is 46.6 mol%.
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