Antonie van Leeuwenhoek DOI 10.1007/s10482-015-0465-8
ORIGINAL PAPER
Actinomadura amylolytica sp. nov. and Actinomadura cellulosilytica sp. nov., isolated from geothermally heated soil Jian-Yu Jiao . Lan Liu . En-Min Zhou . Da-Qiao Wei . Hong Ming . Wen-Dong Xian . Chang-Guo Yuan . Jing-Mei Zhong . Wen-Jun Li
Received: 15 January 2015 / Accepted: 27 April 2015 Ó Springer International Publishing Switzerland 2015
Abstract Two aerobic, Gram-positive actinomycetes, designated YIM 77502T and YIM 77510T, were isolated from geothermally heated soil of Tengchong county, Yunnan province, south-west China. The taxonomic position of strains YIM 77502T and YIM 77510T were investigated by a polyphasic approach. Phylogenetic analyses based on 16S rRNA gene sequences showed that strains YIM 77502T and YIM 77510T belong to the genus Actinomadura. Both strains form extensively-branched substrate and aerial mycelia which differentiated into short spore chains.
Jian-Yu Jiao and Lan Liu have contributed equally to this work.
Electronic supplementary material The online version of this article (doi:10.1007/s10482-015-0465-8) contains supplementary material, which is available to authorized users.
The cell wall of the two strains contained mesodiaminopimelic acid, while the whole-cell sugars detected were glucose, madurose, mannose and rhamnose. The polar lipid profile of strain YIM 77502T was found to consist of diphosphatidylglycerol, phosphatidylinositol mannoside, phosphatidylinositol, two unidentified phospholipids and an unidentified polar lipid, while strain YIM 77510T consisted of diphosphatidylglycerol, phosphatidylinositol mannoside and phosphatidylinositol. The respiratory quinones of strains YIM 77502T and YIM 77510T were MK-9(H6) and MK-9(H8). The major fatty acids ([10 %) of strain YIM 77502T were C17:0, iso-C16:0, C17:010-methyl and iso-C18:0, and those of strain YIM 77510T were iso-C16:0, C17:010-methyl and iso-C18:0. The G?C contents of strains YIM 77502T and YIM 77510T were determined to be 71.3 and
J.-Y. Jiao D.-Q. Wei J.-M. Zhong The Affiliated Hospital of Kunming University of Science and Technology, Kunming University of Science and Technology, Kunming 650550, People’s Republic of China e-mail: [email protected]
L. Liu E.-M. Zhou W.-J. Li (&) State Key Laboratory of Biocontrol and Guangdong Key Laboratory of Plant Resources, College of Ecology and Evolution, Sun Yat-Sen University, Guangzhou 510275, People’s Republic of China e-mail: [email protected]
J.-Y. Jiao L. Liu E.-M. Zhou H. Ming W.-D. Xian C.-G. Yuan W.-J. Li Yunnan Institute of Microbiology, Yunnan University, Kunming 650091, People’s Republic of China
H. Ming College of Life Sciences and Technology, Xinxiang Medical University, Xinxiang 453003, People’s Republic of China
J.-Y. Jiao J.-M. Zhong The First People’s Hospital of Yunnan Province, Kunming 650032, People’s Republic of China
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70.2 mol%, respectively. The DNA–DNA hybridization values of strains YIM 77502T, YIM 77510T and their closest phylogenetic neighbours Actinomadura echinospora BCRC 12547T and Actinomadura umbrina KCTC 9343T were less than 70 %. Based on the morphological and physiological properties, and phylogenetic analyses, strains YIM 77502T and YIM 77510T are considered to represent two novel species of the genus Actinomadura, for which the names Actinomadura amylolytica sp. nov. (type strain YIM 77502T = DSM 45822T = CCTCC AA 2012024T) and Actinomadura cellulosilytica sp. nov. (type strain YIM 77510T = DSM 45823T = CCTCC AA 2012023T) are proposed. Keywords Actinomadura amylolytica sp. nov. Actinomadura cellulosilytica sp. nov. Tengchong Polyphasic taxonomy
Introduction The genus Actinomadura, which belongs to the family Thermomonosporaceae, was first described by Lechevalier and Lechevalier (1970), and subsequently revised by Zhang et al. (2001) and Miyadoh and Miyara (2001). Members of the genus Actinomadura form extensively branched, non-fragmenting substrate mycelia, while their aerial mycelia is either moderately developed or absent. However, when present, aerial mycelia on maturity form short or occasionally long chains of arthrospores with straight, hooked or irregular spiral spore chains, and with folded, irregular, rugose, smooth, spiny or warty spore surfaces. They are also characterized by the presence of mesodiaminopimelic acid and madurose in whole-organism hydrolysates, the predominant menaquinones are MK9(H4), MK-9(H6) and MK-9(H8). Diphosphatidylglycerol and phosphatidylinositol are the major phospholipids (Trujillo and Goodfellow 2012). During the investigation on microbial diversity of geothermally heated soil collected from Tengchong of Yunnan province, south-west China, two strains designated as YIM 77502T and YIM 77510T were isolated. Their 16S rRNA gene sequence similarities were less than 98 % when compared to the 75 validly published species of the genus Actinomadura (http:// www.bacterio.net/actinomadura.html; Parte 2014). The genus Actinobacteria has been previously studied
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for many enzyme activities (Cook et al. 2005; Quintana et al. 2003). However, the ability of the isolates to adapt the high temperature of geothermally heated soil has not been extensively studied. These unique physiological mechanisms inspired the current study to identify the phenotypic, chemotaxonomic and molecular characteristics of these organisms.
Materials and methods Strains and culture conditions Strains YIM 77502T and YIM 77510T were isolated from a geothermally heated soil sample of Tengchong county (Yunnan province, south-west China) on ISP 2 agar medium following serial dilution procedure and incubation at 45 °C for 7–10 days. Pure colonies were obtained by repeatedly streaking on the ISP 2 agar medium and routinely cultivated on the same medium. Both strains were stored as glycerol suspensions (20 %, v/v) at -80 °C for further use. Biomass for chemotaxonomic and molecular studies was obtained by cultivation on ISP 2 agar medium (45 °C, 4 days) except for fatty acids determination. The type strains Actinomadura echinospora BCRC 12547T and Actinomadura umbrina KCTC 9343T were obtained from Bioresource Collection and Research Center (BCRC) and Korean Collection for Type Cultures (KCTC), respectively, and they were cultured under the same conditions. Phenotypic characteristics Gram staining was carried out by using the standard Gram reaction and was confirmed by using the KOH test (Cerny 1978). Cultural characteristics were tested on tryptone yeast agar (ISP 1), yeast extract-malt extract agar (ISP 2), oatmeal agar (ISP 3), inorganic salts-starch agar (ISP 4), glycerol-asparagine agar (ISP 5) (Shirling and Gottlieb 1966), Czapek’s agar (CK), nutrient agar (NA) (Waksman 1967), Gause’s synthetic agar and reasoner0 s 2A agar (R2A). The colours of the colonies were determined by using the ISCC-NBS colour charts (Kelly 1964). The morphological characteristics of strains YIM 77502T and YIM 77510T were determined by light microscopy (Philips XL30) and scanning electron microscopy (ESEMTMP) after the cultures were grown on ISP 2 agar medium at 45 °C for 4 days. Growth at different
Antonie van Leeuwenhoek
temperatures (0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 and 60 °C) and NaCl tolerance at various concentrations (0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4 and 5.0 % w/v) was determined using ISP 2 agar medium. The pH range (4.0–10.0, at intervals of 1.0 pH unit) for growth was tested in ISP 2 broth using buffer system as described by Xu et al. (2005). Oxidase activity was evaluated via the oxidation of tetramethyl-p-phenylenediamine (Kovacs 1956). Catalase activity was detected by assessing the production of bubbles on addition of a drop of 3 % (v/v) H2O2. H2S production, milk peptonization and coagulation, nitrate reduction, urease activity and hydrolysis of cellulose, gelatin, starch and Tweens (20, 40, 60 and 80) were performed as described by Gonzalez et al. (1978). Sole carbon source utilization was determined by the methods as described by Shirling and Gottlieb (1966) and Locci (1989), while sole nitrogen source utilization was examined according to Williams et al. (1989). Antibiotic susceptibility test was performed by the agar-diffusion method using 0.5 McFarland bacterial suspensions plated onto ISP 2 agar medium (45 °C, 4 days). The antimicrobial susceptibility was determined by measuring the zone of inhibition. Chemotaxonomy The isomer of amino acids in purified cell-wall and sugars of whole-cell hydrolysates were determined according to the procedures described by Hasegawa et al. (1983), Staneck and Roberts (1974) and Tang et al. (2009). Polar lipids were prepared as described by Minnikin et al. (1979) and identified by twodimensional TLC (Collins and Jones 1980). Menaquinones were extracted (Collins et al. 1977) and analyzed using HPLC (Kroppenstedt 1982). Cellular fatty acid analysis was performed by using the Microbial Identification System (Sherlock Version 6.1; MIDI database: TSBA6; Sasser 1990). Biomass for fatty acid analysis was obtained from cells grown in Tryptic Soy Broth (TSB) at 45 °C for 4 days. The G?C content of the genomic DNAs were determined by using reversed-phase HPLC (Mesbah et al. 1989) with Escherichia coli DH5a as the reference strain. Molecular analysis and DNA–DNA hybridization Extraction of genomic DNAs and PCR amplification of the 16S rRNA genes were performed as described
by Li et al. (2007). The sequences obtained were compared with available 16S rRNA gene sequences of validly published species from the EzTaxon-e server (http://eztaxon-e.ezbiocloud.net/; Kim et al. 2012). Multiple alignments with sequences of the most closely related taxa were carried out by using CLUSTAL_X program (Thompson et al. 1997). Phylogenetic analyses were performed by using three treemaking algorithms: neighbour-joining (Saitou and Nei 1987), maximum-likelihood (Felsenstein 1981) and maximum-parsimony (Fitch 1971). The trees were constructed by using the MEGA version 5.0 software package (Tamura et al. 2011). Kimura’s two parameter model was used to calculate evolutionary distance matrices of the phylogenetic trees (Kimura 1980). Bootstrap analysis was performed with 1000 replications (Felsenstein 1985). Streptomyces glauciniger FXJ14T (AY314782) was used as an outgroup. DNA–DNA relatedness was studied using the fluorometric micro-well method (Ezaki et al. 1989; Christensen et al. 2000) at 50 °C as the optimal hybridization temperature. The experiments were set with eight replications between strains YIM 77502T, YIM 77510T and their closest phylogenetic neighbours A. echinospora BCRC 12547T and A. umbrina KCTC 9343T.
Results and discussion Phenotypic characteristics Cells of strains YIM 77502T and YIM 77510T were observed to be Gram-staining positive, aerobic and non-motile. Strains YIM 77502T and YIM 77510T produced abundantly branched, non-fragmenting substrate and aerial mycelia, which differentiated into spore chains bearing rugose spores (Fig. S1). Three weeks old cultures of strains YIM 77502T and YIM 77510T revealed that white aerial mycelia were produced on all the tested media. Strain YIM 77502T grew well on Czapek’s agar, Gause’s synthetic agar, ISP 2 and R2A, grew weakly on ISP 1, ISP 3, ISP4, ISP 5 and NA. The substrate mycelia were observed to be either yellow–white or orange–yellow colour. Diffusible pigment (Grayish yellowish-brown) was produced on Gause’s synthetic agar. Strain YIM 77510T grew well on ISP 1, ISP 2, ISP 3, ISP 4, Gause’s synthetic agar, NA and R2A, grew weakly on ISP 5
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and Czapek’s agar. The colours of substrate mycelia range within orange-yellow, yellowish-brown, yellow or yellow-white, and no diffusible pigment was produced (Table S1). Both strains can grow over a wide range of temperature (25–55 °C) and can tolerate NaCl up to 3 % (w/v). However, the two strains were found to differ in their pH range for growth. Strain YIM 77502T was found to grow at a pH range of 6.0–9.0 with an optimum pH 7.0, while strain YIM 77510T had a pH range of 7.0–8.0 with an optimum pH 7.0. Both strains were positive for catalase, milk peptonization, nitrate reduction, urease and hydrolysis of cellulose and Tween 40 but negative for oxidase, H2S production and hydrolysis of Tweens (20, 60 and 80). Strain YIM 77502T showed positive result for milk coagulation and starch hydrolysis but negative result for gelatin liquefaction, while strain YIM 77510T was found to be positive for gelatin liquefaction and negative for milk coagulation and starch hydrolysis tests. Strains YIM 77502T and YIM 77510T were sensitive to amikacin (30 lg), chloramphenicol (30 lg), ciprofloxacin (5 lg), erythromycin (15 lg), gentamicin (10 lg), kanamycin (30 lg), natilmicin (30 lg), novobiocin (5 lg), polymyxin B (300U), streptomycin (10 lg), tetracycline (30 lg), tobramycin (10 lg) and vancomycin (30 lg). The only difference between the two strains was that strain YIM 77502T was sensitive to penicillin G (10 IU) but not strain YIM 77510T. The major differential characteristics between strains YIM 77502T, YIM 77510T and the related type strains A. echinospora BCRC 12547T and A. umbrina KCTC 9343T are shown in Table 1. The detailed physiological characteristics of strains YIM 77502T and YIM 77510T are given in the species description. Chemotaxonomy The cell wall of the strains YIM 77502T and YIM 77510T contained meso-diaminopimelic acid, while the whole-cell sugars were glucose, madurose, mannose and rhamnose. The polar lipid profile of strain YIM 77502T was found to consist of diphosphatidylglycerol, phosphatidylinositol mannoside, phosphatidylinositol, two unidentified phospholipids and an unidentified polar lipid, while strain YIM 77510T consisted of diphosphatidylglycerol, phosphatidylinositol mannoside and phosphatidylinositol. The respiratory quinone for both strains were MK-9(H6) and
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MK-9(H8). The major fatty acids ([10 %) of strain YIM 77502T were C17:0, iso-C16:0, C17:0 10-methyl, iso-C18:0, while those of strain YIM 77510T were isoC16:0, C17:0 10-methyl, iso-C18:0, which is consistent with the profiles described for other members of the genus Actinomadura, though their proportions were different (Table 2). The G?C contents of strains YIM 77502T and YIM 77510T were determined to be 71.3 and 70.2 mol%, respectively. Phylogenetic analysis and DNA–DNA relatedness The almost complete 16S rRNA gene sequences of strains YIM 77502T (1540 bp) and YIM 77510T (1545 bp) were obtained. The GenBank accession numbers are AB859253 and AB859254, respectively. Sequence analysis of the almost complete 16S rRNA gene sequences of the two strains using the EzTaxon-e server showed that they had highest similarities to members of the genus Actinomadura, and are closely related to A. echinospora BCRC 12547T (97.6 and 97.3 % similarity) and A. umbrina KCTC 9343T (97.4 and 98.0 %). The two strains had 98.4 % similarity between them. The neighbour-joining phylogenetic tree based on 16S rRNA sequences showed the two strains form a clade with A. echinospora BCRC 12547T and A. umbrina KCTC 9343T, and were well separated from other members of the genus Actinomadura (Fig. 1). The stabilities of trees were further confirmed by maximum-likelihood and maximumparsimony methods (Figs. S3 and S4). The levels of DNA–DNA relatedness of strain YIM 77502T with A. echinospora BCRC 12547T and A. umbrina KCTC 9343T were 56.1 ± 2.5 and 44.9 ± 1.9 %, respectively, while the DNA–DNA hybridization values for strain YIM 77510T with respect to A. echinospora BCRC 12547T and A. umbrina KCTC 9343T were 10.2 ± 2.4 and 28.1 ± 3.4 %, respectively. The two strains YIM 77502T and YIM 77510T also had a DNA–DNA relatedness value of 36.9 ± 3.8 %, thereby indicating that all the DNA–DNA hybridization values were less than the cut-off point (70 %) for the delineation of genomic species (Stackebrandt and Goebel 1994). Therefore, on the basis of phylogenetic analysis, phenotypic and chemotaxonomic characteristics (Tables 1, 2; Fig. 1, Figs S1–S4), strains YIM 77502T and YIM 77510T should be affiliated to the genus Actinomadura. Strains YIM 77502T and YIM
Antonie van Leeuwenhoek Table 1 Different characteristics among strains YIM 77502T, YIM 77510T, A. echinospora BCRC 12547T and A. umbrina KCTC 9343T
Characteristics
YIM 77502T
YIM 77510T
BCRC 12547T
KCTC 9343T
Optimum temperature
45
45
35
30
NaCl tolerance(%)
3
3
2.5
2.5
Milk peptonization
?
?
-
-
Milk coagulation
?
-
-
-
Nitrate reduction
?
?
?
-
Tween 40
?
?
-
?
Starch Gelatin
? -
?
? ?
-
Cellulose
?
?
-
-
D-galactose
?
?
-
-
D-mannose
-
-
?
?
D-sorbitol
-
?
-
-
Glucose
?
?
-
?
Inositol
?
-
-
?
L-rhamnose
?
-
?
?
Mannitol
-
?
-
-
Xylitol
?
-
-
?
Arginine
?
-
?
-
Aspartic acid
?
-
?
-
DL-methionine
-
? -
? ?
-
Degradation of
Carbon source utilization
Nitrogen source utilization
Glycine
All the data were obtained from this study ?, positive, - negative
Hypoxanthine
?
-
-
-
L-histidine
-
-
?
-
L-threonine
?
?
?
-
L-tyrosine
?
?
?
-
L-valine
?
?
?
-
Lysine
?
-
?
-
Phenylalanine
-
-
?
-
Proline
?
?
?
-
Tryptophan
?
-
?
?
Xanthine
?
-
-
-
?
-
?
?
Sensitive to PenicillinG
77510T could be distinguished from the type strains A. echinospora BCRC 12547T and A. umbrina KCTC 9343T by several properties, such as temperature range and NaCl tolerance for growth, hydrolysis of cellulose, gelatin, starch, and Tween 40, utilization of carbon sources and nitrogen sources, and the proportions of some fatty acids. In addition, analyses of 16S rRNA gene sequences, DNA–DNA relatedness
values and their different properties, especially enzyme activities, the antibiotic sensitivity and utilization of sole-carbon sources and nitrogen sources, indicate that strains YIM 77502T and YIM 77510T represent two novel species within the genus Actinomadura, for which the names Actinomadura amylolytica sp. nov. and Actinomadura cellulosilytica sp. nov. are proposed, respectively.
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Antonie van Leeuwenhoek Table 2 Cellular fatty acid profiles (% of total) for strains YIM 77502T, YIM 77510T, Actinomadura echinospora BCRC 12547T and Actinomadura umbrina KCTC 9343T
Fatty acid (%)
YIM 77502T
YIM 77510T
BCRC 12547T
KCTC 9343T
Saturated fatty acids C16:0
2.8
3.9
4.8
4.7
C17:0
16.3
6.6
8.2
2.5
C18:0
2.2
5.0
3.7
6.3
C19:0
1.9
2.1
1.1
0.3
Branched fatty acids
All data were obtained in this study. Values are percentage of total fatty acid. ND not detected, Tr trace, mean values less than 0.5 %. Fatty acids that represent \0.5 % in all strains were omitted a
Summed in feature nine comprises 10-methyl C16:0; summed feature nine comprised iso-C17:1 x9c and/or 10-methyl C16:0
Iso-C16:0
37.5
26.3
42.7
46.1
C17:0 10-methyl Iso-C18:0
19.4 10.9
13.0 16.8
8.4 17.1
12.0 13.5
Iso-C14:0
1.0
0.5
1.9
0.9
Anteiso-C15:0
Tr
Tr
Tr
0.6
Iso-C15:0
Tr
0.8
ND
Tr
Anteiso-C17:0
1.6
4.1
0.7
0.6
Iso-C17:0
1.3
6.1
ND
Tr
Anteiso-C19:0
ND
1.0
ND
ND
Iso-C19:0
ND
0.5
ND
ND
C18:0 10-methyl
1.1
7.4
2.8
6.5
C19:0 10-methyl
ND
Tr
Tr
1.0
Unsaturated fatty acids C17:1x8c
2.0
1.5
3.7
Tr
C18:1x9c
Tr
0.8
1.4
0.5
Iso G-C16:1
ND
0.6
Tr
Tr
Tr
1.0
Tr
1.3
Sum In Feature 9a
Description of Actinomadura amylolytica sp. nov Actinomadura amylolytica (a.my.lo.ly0 ti.ca. Gr. n. amulon, starch; Gr. adj. lutikos, dissolving; N. L. fem. adj. amylolytica starch dissolving). Aerobic, Gram-staining positive; forms extensively branched, non-fragmenting, orange-yellow or yellowwhite substrate mycelia; bear white aerial mycelia on all the tested media. Aerial mycelia produce spiral spore chains with rugose surface. Diffusible pigments are produced on Gause’s synthetic agar. Growth occurs at 25–55 °C and pH 6.0–9.0 with optimal growth at 45 °C and pH 7.0. Can tolerate 0–3 % (w/v) NaCl. Positive for catalase, milk peptonization and coagulation, nitrate reduction, urease and cellulose, starch and Tween 40 hydrolysis tests, but negative for oxidase, H2S production and hydrolysis of gelatin and Tweens 20, 60 and 80. Utilizes D-fructose, D-galactose, glucose, inositol, L-rhamnose and xylitol as the sole carbon and energy sources, but not arabinose,
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cellose, a-lactose, maltose, mannitol, D-mannose, raffinose, sodium malate, D-sorbitol, succinic acid, sucrose, trehalose and xylose as the sole carbon sources. Alanine, arginine, aspartic acid, hypoxanthine, lysine, proline, serine, L-threonine, tryptophan, L-tyrosine, L-valine and xanthine can be used as the sole nitrogen sources, but not glutamic acid, glycine, L-histidine, DL-methionine or phenylalanine. The cell wall contains meso-DAP, whole-cell sugars contain glucose, madurose, mannose and rhamnose. The polar lipid profile consists of diphosphatidylglycerol, phosphatidylinositol mannoside, phosphatidylinositol, two unidentified phospholipids and an unidentified polar lipid. MK-9(H6) and MK-9(H8) are the predominant menaquinones. The major fatty acids ([10 %) are C17:0, iso-C16:0, C17:0 10-methyl, iso-C18:0. The G?C content of the type strain is 71.3 mol%. The type strain YIM 77502T (=DSM 45822T = CCTCC AA 2012024T) was isolated from the geothermally heated soil located in Tengchong
Antonie van Leeuwenhoek Fig. 1 Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences of strains YIM 77502T, YIM 77510T and their closest relatives. Bootstrap values (expressed as percentages of 1000 replications) above 50 % are shown at the branch points. Asterisks denote nodes that were also recovered using the maximum-parsimony and maximum-likelihood methods. The sequence of Streptomyces glauciniger FXJ14T was used as the outgroup. Bar 0.01, represents substitutions per nucleotide position
81* Actinomadura verrucosospora NBRC 14100T/U49011 66* Actinomadura coerulea IFO 14679T/U49002 97* Actinomadura citrea IFO 14678T/U49001 74* Actinomadura mexicana A290T/AF277195 Actinomadura xylanilytica BK147T/FR692101 Actinomadura bangladeshensis 3-46-b3T/AB331652 99* Actinomadura chokoriensis 3-45-a/11T/AB331730 93* Actinomadura geliboluensis A8036T/HQ157187 76* Actinomadura meyerae A288T/AY273787
*
Actinomadura yumaensis JCM 3369T/AF163122 Actinomadura chibensis IFM 10266T/AB264086
* * 62
Actinomadura livida JCM 3387T/AF163116 Actinomadura vinacea JCM 3325T/AF134070 99* Actinomadura viridis IFO 15238T/D85467
99* 56*
60* 85*
*
60 80
Actinomadura sputiIMMIB L-889T/FM957483 Actinomadura hallensis H647-1T/DQ076484 Actinomadura miaoliensis BC 44T-5T/EF116925 100* Actinomadura keratinilytica WCC-2265T/EU637009 Actinomadura atramentaria IFO 14695T/U49000 Actinomadura flavalba YIM 61435T/FJ157185 Actinomadura echinospora IFO 14042T/U49004 Actinomadura umbrina JCM 6837T/AF163121 Actinomadura cellulosilytica YIM 77510T/AB859254 Actinomadura amylolytica YIM 77502T/AB859253 Thermomonospora curvata DSM 43183T/CP001738 Streptomyces glauciniger FXJ14T/AY314782
0.01
county, Yunnan province, south-west China. The GenBank accession number for the 16S rRNA gene sequence of strain YIM 77502T is AB859253. Description of Actinomadura cellulosilytica sp. nov Actinomadura cellulosilytica (cel.lu.lo.si.ly’ ti.ca. N.L. n. cellulosum cellulose; N.L. adj. lyticus dissolving; N.L. fem. adj. cellulosilytica cellulose-dissolving). Aerobic, Gram-staining positive. Forms extensively branched, non-fragmenting, orange–yellow, yellowish–brown, yellow or yellow–white substrate mycelia; aerial mycelia differentiate into chains of spores with rugose surface. Diffusible pigments are not produced on any of the tested media. Growth occurs at 25–55 °C and pH 7.0–8.0 with optimal growth at 45 °C and at pH 7.0. Can tolerate up to 3 % (w/v) NaCl. Cellulose, gelatin, Tween 40 and urea are hydrolysed, but not Tweens (20, 60 and 80). Positive for catalase, milk peptonization and nitrate reduction but negative for milk coagulation, oxidase, H2S production and starch
hydrolysis tests. Utilizes D-fructose, D-galactose, glucose, mannitol and D-sorbitol as the sole carbon and energy sources, but not arabinose, cellulose, inositol, a-lactose, maltose, D-mannose, raffinose, L-rhamnose, sodium malate, succinic acid, sucrose, trehalose, xylitol and xylose as the sole carbon sources. Alanine, DL-methionine, proline, serine, L-threonine, L-tyrosine and L-valine can be used as the sole nitrogen sources, but not arginine, aspartic acid, glutamic acid, glycine, L-histidine, hypoxanthine, lysine, phenylalanine, tryptophan and xanthine. The cell wall contains mesoDAP, whole-cell sugars contain glucose, madurose, mannose and rhamnose. The polar lipids profile consists of diphosphatidylglycerol, phosphatidylinositol mannoside and phosphatidylinositol. The respiratory quinones are MK-9(H6) and MK-9(H8). The major fatty acids ([10 %) are iso-C16:0, C17:0 10-methy and iso-C18:0, The G?C content of the type strain is 70.2 mol%. The type strain YIM 77510T (=DSM 45823T = CCTCC AA 2012023T) was isolated from the geothermally heated soil located in Tengchong
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county, Yunnan province, south-west China. The GenBank accession number for the 16S rRNA gene sequence of strain YIM 77510T is AB859254. Acknowledgments We are grateful to Dr. Jung-Sook Lee (KCTC) and Dr. Min Tseng (BCRC) for their kind providing reference type strains, and Prof. Aharon Oren (The Hebrew University of Jerusalem, Israel) for his kind help with the Latin etymology for the new species and Dr. Nimaichand Salam (Manipur University, India) for his help to further polishing this manuscript. This research was supported by Key Project of International Cooperation of Ministry of Science & Technology (MOST) (No. 2013DFA31980), Natural Science Foundation of China (No. 31470139) and Yunnan Provincial Natural Science Foundation (2013FA004). W-J Li was also supported by Guangdong Province Higher Vocational Colleges & Schools Pearl River Scholar Funded Scheme (2014).
References Cerny G (1978) Studies on aminopeptidase for the distinction of gram-negative from gram-positive bacteria. Appl Microbiol Biotechnol 5:113–122 Christensen H, Angen O, Mutters R, Olsen JE, Bisgaard M (2000) DNA-DNA hybridization determined in microwells using covalent attachment of DNA. Int J Syst Evol Microbiol 50:1095–1102 Collins MD, Jones D (1980) Lipids in the classification and identification of coryneform bacteria containing peptidoglycan based on 2, 4-diaminobutyric acid. J Appl Bacteriol 48:459–470 Collins MD, Pirouz T, Goodfellow M, Minnikin DE (1977) Distribution of menaquinones in actinomycetes and corynebacteria. J Gen Microbiol 100:221–230 Cook AE, Marilize LR, Meyers PR (2005) Actinomadura napierensis sp. Nov., isolated from soil in South Africa. Int J Syst Evol Microbiol 55:703–706 Ezaki T, Hashimoto Y, Yabuuchi E (1989) Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39:224–229 Felsenstein J (1981) Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol 17:368–376 Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–790 Fitch WM (1971) To ward defining the course of evolution: minimum change for a specific tree topology. Syst Zool 20:406–416 Gonzalez C, Gutierrez C, Ramirez C (1978) Halobacterium vallismortis sp. nov., an amylolytic and carbohydrate-metabolizing, extremely halophilic bacterium. Can J Microbiol 24:710–715 Hasegawa T, Takizawa M, Tanida S (1983) A rapid analysis for chemical grouping of aerobic actinomycetes. J Gen Microbiol 29:319–322
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Kelly KL (1964) Inter-Society Color Council-National Bureau of Standards Color Name charts illustrated with centroid colors. US Government Printing Office, Washington Kim OS, Cho YJ, Lee K, Yoon SH, Kim M, Na H, Park SC, Jeon YS, Lee JH, Yi H, Won S, Chun J (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 Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16:111–120 Kovacs N (1956) Identification of Pseudomonas pyocyanea by the oxidase reaction. Nature 178:703–704 Kroppenstedt RM (1982) Separation of bacterial menaquinones by HPLC using reverse phase (RP18) and a silver loadedion exchanger as stationary phases. J Liq Chromatogr 5:2359–2367 Lechevalier MP, Lechevalier HA (1970) Chemical composition as a criterion in the classification of aerobic actinomycetes. Int J Syst Bacteriol 20:435–443 Li WJ, Xu P, Schumann P, Zhang YQ, Pukall R, Xu LH, Stackebrandt E, Jiang CL (2007) Georgenia ruanii sp. nov., a novel actinobacterium isolated from forest soil in Yunnan (China) and emended description of the genus Georgenia. Int J Syst Evol Microbiol 57:1424–1428 Locci R (1989) Streptomyces and related genera. In: Williams ST, Sharpe ME, Holt JG (eds) Bergey’s manual of systematic bacteriology, vol 4. Williams & Wilkins, Baltimore, pp 2451–2508 Mesbah M, Premachandran U, Whitman WB (1989) Precise measurement of the G?C content of deoxyribonucleic acidby high-performance liquid chromatography. Int J Syst Bacteriol 39:159–167 Minnikin DE, Collins MD, Goodfellow M (1979) Fatty acid and polar lipid composition in the classification of Cellulomonas, Oerskovia and related taxa. J Appl Bacteriol 47:87–95 Miyadoh S, Miyara T (2001) Family Thermomonosporaceae. In: The Society for Actinomycetes (ed) Identification manual of actinomycetes. Business Center for Academic Societies, Tokyo, pp 281–291 Parte AC (2014) LPSN—list of prokaryotic names with standing in nomenclature. Nucleic Acids Res 42:D613–D616 Quintana ET, Trujillo ME, Goodfellow M (2003) Actinomadura mexicana sp. nov. and Actinomadura meyerii sp. nov., two novel soil sporoactinomycetes. Syst Appl Microbiol 26:511–517 Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425 Sasser M (1990) Identification of bacteria by gas chromatography of cellular fatty acids. USFCC Newsl 20:16 Shirling EB, Gottlieb D (1966) Methods for characterization of Streptomyces species. Int J Syst Bacteriol 16:313–340 Stackebrandt E, Goebel BM (1994) Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44:846–849 Staneck JL, Roberts GD (1974) Simplified approached to identification of aerobic actinomycetes by thin-layer chromatography. Appl Mirobiol 28:2731–2739
Antonie van Leeuwenhoek Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739 Tang SK, Wang Y, Chen Y, Lou K, Cao LL, Xu LH, Li WJ (2009) Zhihengliuella alba sp. nov., and emended description of the genus Zhihengliuella. Int J Syst Evol Microbiol 59:2025–2032 Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTALX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882 Trujillo ME, Goodfellow M (2012) Genus III. Actinomadura Lechevalier and lechevalier 1970, 400AL emend. Kroppenstedt, Stackebrandt and Goodfellow 1990, 156. In: Goodfellow M, Ka¨mpfer P, Busse MJ, Trujillo ME, Suzuki KL, Ludwig W, Whitman WB (eds) Bergey’s Manual of Systematic Bacteriology, vol 5, 2nd edn. Springer, New York, pp 1940–1959
Waksman SA (1967) The actinomycetes: a summary of current knowledge. Ronald Press, New York Williams ST, Goodfellow M, Alderson G (1989) Genus StreptomycesWaksman and Henrici 1943, 339AL. In: Williams ST, Sharpe ME, Holt JG (eds) Bergey’s manual of systematic bacteriology, vol 4. Williams and Willkins, Baltimore, pp 2453–2492 Xu P, Li WJ, Tang SK, Zhang YQ, Chen GZ, Chen HH, Xu H, Jiang CL (2005) Naxibacter alkalitolerans gen. nov., sp. nov., a novel member of the family Oxalobacteraceae isolated from China. Int J Syst Evol Microbiol 55:1149–1153 Zhang Z, Kudo T, Nakajima Y, Wang Y (2001) Clarification of the relationship between the members of the family Thermomonosporaceae on the basis of 16S rDNA, 16S-23S rRNA internal transcribed spacer and 23S rDNA sequences and chemotaxonomic analyses. Int J Syst Evol Microbiol 51:373–383
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ORIGINAL PAPER
Actinomadura amylolytica sp. nov. and Actinomadura cellulosilytica sp. nov., isolated from geothermally heated soil Jian-Yu Jiao . Lan Liu . En-Min Zhou . Da-Qiao Wei . Hong Ming . Wen-Dong Xian . Chang-Guo Yuan . Jing-Mei Zhong . Wen-Jun Li
Received: 15 January 2015 / Accepted: 27 April 2015 Ó Springer International Publishing Switzerland 2015
Abstract Two aerobic, Gram-positive actinomycetes, designated YIM 77502T and YIM 77510T, were isolated from geothermally heated soil of Tengchong county, Yunnan province, south-west China. The taxonomic position of strains YIM 77502T and YIM 77510T were investigated by a polyphasic approach. Phylogenetic analyses based on 16S rRNA gene sequences showed that strains YIM 77502T and YIM 77510T belong to the genus Actinomadura. Both strains form extensively-branched substrate and aerial mycelia which differentiated into short spore chains.
Jian-Yu Jiao and Lan Liu have contributed equally to this work.
Electronic supplementary material The online version of this article (doi:10.1007/s10482-015-0465-8) contains supplementary material, which is available to authorized users.
The cell wall of the two strains contained mesodiaminopimelic acid, while the whole-cell sugars detected were glucose, madurose, mannose and rhamnose. The polar lipid profile of strain YIM 77502T was found to consist of diphosphatidylglycerol, phosphatidylinositol mannoside, phosphatidylinositol, two unidentified phospholipids and an unidentified polar lipid, while strain YIM 77510T consisted of diphosphatidylglycerol, phosphatidylinositol mannoside and phosphatidylinositol. The respiratory quinones of strains YIM 77502T and YIM 77510T were MK-9(H6) and MK-9(H8). The major fatty acids ([10 %) of strain YIM 77502T were C17:0, iso-C16:0, C17:010-methyl and iso-C18:0, and those of strain YIM 77510T were iso-C16:0, C17:010-methyl and iso-C18:0. The G?C contents of strains YIM 77502T and YIM 77510T were determined to be 71.3 and
J.-Y. Jiao D.-Q. Wei J.-M. Zhong The Affiliated Hospital of Kunming University of Science and Technology, Kunming University of Science and Technology, Kunming 650550, People’s Republic of China e-mail: [email protected]
L. Liu E.-M. Zhou W.-J. Li (&) State Key Laboratory of Biocontrol and Guangdong Key Laboratory of Plant Resources, College of Ecology and Evolution, Sun Yat-Sen University, Guangzhou 510275, People’s Republic of China e-mail: [email protected]
J.-Y. Jiao L. Liu E.-M. Zhou H. Ming W.-D. Xian C.-G. Yuan W.-J. Li Yunnan Institute of Microbiology, Yunnan University, Kunming 650091, People’s Republic of China
H. Ming College of Life Sciences and Technology, Xinxiang Medical University, Xinxiang 453003, People’s Republic of China
J.-Y. Jiao J.-M. Zhong The First People’s Hospital of Yunnan Province, Kunming 650032, People’s Republic of China
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70.2 mol%, respectively. The DNA–DNA hybridization values of strains YIM 77502T, YIM 77510T and their closest phylogenetic neighbours Actinomadura echinospora BCRC 12547T and Actinomadura umbrina KCTC 9343T were less than 70 %. Based on the morphological and physiological properties, and phylogenetic analyses, strains YIM 77502T and YIM 77510T are considered to represent two novel species of the genus Actinomadura, for which the names Actinomadura amylolytica sp. nov. (type strain YIM 77502T = DSM 45822T = CCTCC AA 2012024T) and Actinomadura cellulosilytica sp. nov. (type strain YIM 77510T = DSM 45823T = CCTCC AA 2012023T) are proposed. Keywords Actinomadura amylolytica sp. nov. Actinomadura cellulosilytica sp. nov. Tengchong Polyphasic taxonomy
Introduction The genus Actinomadura, which belongs to the family Thermomonosporaceae, was first described by Lechevalier and Lechevalier (1970), and subsequently revised by Zhang et al. (2001) and Miyadoh and Miyara (2001). Members of the genus Actinomadura form extensively branched, non-fragmenting substrate mycelia, while their aerial mycelia is either moderately developed or absent. However, when present, aerial mycelia on maturity form short or occasionally long chains of arthrospores with straight, hooked or irregular spiral spore chains, and with folded, irregular, rugose, smooth, spiny or warty spore surfaces. They are also characterized by the presence of mesodiaminopimelic acid and madurose in whole-organism hydrolysates, the predominant menaquinones are MK9(H4), MK-9(H6) and MK-9(H8). Diphosphatidylglycerol and phosphatidylinositol are the major phospholipids (Trujillo and Goodfellow 2012). During the investigation on microbial diversity of geothermally heated soil collected from Tengchong of Yunnan province, south-west China, two strains designated as YIM 77502T and YIM 77510T were isolated. Their 16S rRNA gene sequence similarities were less than 98 % when compared to the 75 validly published species of the genus Actinomadura (http:// www.bacterio.net/actinomadura.html; Parte 2014). The genus Actinobacteria has been previously studied
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for many enzyme activities (Cook et al. 2005; Quintana et al. 2003). However, the ability of the isolates to adapt the high temperature of geothermally heated soil has not been extensively studied. These unique physiological mechanisms inspired the current study to identify the phenotypic, chemotaxonomic and molecular characteristics of these organisms.
Materials and methods Strains and culture conditions Strains YIM 77502T and YIM 77510T were isolated from a geothermally heated soil sample of Tengchong county (Yunnan province, south-west China) on ISP 2 agar medium following serial dilution procedure and incubation at 45 °C for 7–10 days. Pure colonies were obtained by repeatedly streaking on the ISP 2 agar medium and routinely cultivated on the same medium. Both strains were stored as glycerol suspensions (20 %, v/v) at -80 °C for further use. Biomass for chemotaxonomic and molecular studies was obtained by cultivation on ISP 2 agar medium (45 °C, 4 days) except for fatty acids determination. The type strains Actinomadura echinospora BCRC 12547T and Actinomadura umbrina KCTC 9343T were obtained from Bioresource Collection and Research Center (BCRC) and Korean Collection for Type Cultures (KCTC), respectively, and they were cultured under the same conditions. Phenotypic characteristics Gram staining was carried out by using the standard Gram reaction and was confirmed by using the KOH test (Cerny 1978). Cultural characteristics were tested on tryptone yeast agar (ISP 1), yeast extract-malt extract agar (ISP 2), oatmeal agar (ISP 3), inorganic salts-starch agar (ISP 4), glycerol-asparagine agar (ISP 5) (Shirling and Gottlieb 1966), Czapek’s agar (CK), nutrient agar (NA) (Waksman 1967), Gause’s synthetic agar and reasoner0 s 2A agar (R2A). The colours of the colonies were determined by using the ISCC-NBS colour charts (Kelly 1964). The morphological characteristics of strains YIM 77502T and YIM 77510T were determined by light microscopy (Philips XL30) and scanning electron microscopy (ESEMTMP) after the cultures were grown on ISP 2 agar medium at 45 °C for 4 days. Growth at different
Antonie van Leeuwenhoek
temperatures (0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 and 60 °C) and NaCl tolerance at various concentrations (0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4 and 5.0 % w/v) was determined using ISP 2 agar medium. The pH range (4.0–10.0, at intervals of 1.0 pH unit) for growth was tested in ISP 2 broth using buffer system as described by Xu et al. (2005). Oxidase activity was evaluated via the oxidation of tetramethyl-p-phenylenediamine (Kovacs 1956). Catalase activity was detected by assessing the production of bubbles on addition of a drop of 3 % (v/v) H2O2. H2S production, milk peptonization and coagulation, nitrate reduction, urease activity and hydrolysis of cellulose, gelatin, starch and Tweens (20, 40, 60 and 80) were performed as described by Gonzalez et al. (1978). Sole carbon source utilization was determined by the methods as described by Shirling and Gottlieb (1966) and Locci (1989), while sole nitrogen source utilization was examined according to Williams et al. (1989). Antibiotic susceptibility test was performed by the agar-diffusion method using 0.5 McFarland bacterial suspensions plated onto ISP 2 agar medium (45 °C, 4 days). The antimicrobial susceptibility was determined by measuring the zone of inhibition. Chemotaxonomy The isomer of amino acids in purified cell-wall and sugars of whole-cell hydrolysates were determined according to the procedures described by Hasegawa et al. (1983), Staneck and Roberts (1974) and Tang et al. (2009). Polar lipids were prepared as described by Minnikin et al. (1979) and identified by twodimensional TLC (Collins and Jones 1980). Menaquinones were extracted (Collins et al. 1977) and analyzed using HPLC (Kroppenstedt 1982). Cellular fatty acid analysis was performed by using the Microbial Identification System (Sherlock Version 6.1; MIDI database: TSBA6; Sasser 1990). Biomass for fatty acid analysis was obtained from cells grown in Tryptic Soy Broth (TSB) at 45 °C for 4 days. The G?C content of the genomic DNAs were determined by using reversed-phase HPLC (Mesbah et al. 1989) with Escherichia coli DH5a as the reference strain. Molecular analysis and DNA–DNA hybridization Extraction of genomic DNAs and PCR amplification of the 16S rRNA genes were performed as described
by Li et al. (2007). The sequences obtained were compared with available 16S rRNA gene sequences of validly published species from the EzTaxon-e server (http://eztaxon-e.ezbiocloud.net/; Kim et al. 2012). Multiple alignments with sequences of the most closely related taxa were carried out by using CLUSTAL_X program (Thompson et al. 1997). Phylogenetic analyses were performed by using three treemaking algorithms: neighbour-joining (Saitou and Nei 1987), maximum-likelihood (Felsenstein 1981) and maximum-parsimony (Fitch 1971). The trees were constructed by using the MEGA version 5.0 software package (Tamura et al. 2011). Kimura’s two parameter model was used to calculate evolutionary distance matrices of the phylogenetic trees (Kimura 1980). Bootstrap analysis was performed with 1000 replications (Felsenstein 1985). Streptomyces glauciniger FXJ14T (AY314782) was used as an outgroup. DNA–DNA relatedness was studied using the fluorometric micro-well method (Ezaki et al. 1989; Christensen et al. 2000) at 50 °C as the optimal hybridization temperature. The experiments were set with eight replications between strains YIM 77502T, YIM 77510T and their closest phylogenetic neighbours A. echinospora BCRC 12547T and A. umbrina KCTC 9343T.
Results and discussion Phenotypic characteristics Cells of strains YIM 77502T and YIM 77510T were observed to be Gram-staining positive, aerobic and non-motile. Strains YIM 77502T and YIM 77510T produced abundantly branched, non-fragmenting substrate and aerial mycelia, which differentiated into spore chains bearing rugose spores (Fig. S1). Three weeks old cultures of strains YIM 77502T and YIM 77510T revealed that white aerial mycelia were produced on all the tested media. Strain YIM 77502T grew well on Czapek’s agar, Gause’s synthetic agar, ISP 2 and R2A, grew weakly on ISP 1, ISP 3, ISP4, ISP 5 and NA. The substrate mycelia were observed to be either yellow–white or orange–yellow colour. Diffusible pigment (Grayish yellowish-brown) was produced on Gause’s synthetic agar. Strain YIM 77510T grew well on ISP 1, ISP 2, ISP 3, ISP 4, Gause’s synthetic agar, NA and R2A, grew weakly on ISP 5
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and Czapek’s agar. The colours of substrate mycelia range within orange-yellow, yellowish-brown, yellow or yellow-white, and no diffusible pigment was produced (Table S1). Both strains can grow over a wide range of temperature (25–55 °C) and can tolerate NaCl up to 3 % (w/v). However, the two strains were found to differ in their pH range for growth. Strain YIM 77502T was found to grow at a pH range of 6.0–9.0 with an optimum pH 7.0, while strain YIM 77510T had a pH range of 7.0–8.0 with an optimum pH 7.0. Both strains were positive for catalase, milk peptonization, nitrate reduction, urease and hydrolysis of cellulose and Tween 40 but negative for oxidase, H2S production and hydrolysis of Tweens (20, 60 and 80). Strain YIM 77502T showed positive result for milk coagulation and starch hydrolysis but negative result for gelatin liquefaction, while strain YIM 77510T was found to be positive for gelatin liquefaction and negative for milk coagulation and starch hydrolysis tests. Strains YIM 77502T and YIM 77510T were sensitive to amikacin (30 lg), chloramphenicol (30 lg), ciprofloxacin (5 lg), erythromycin (15 lg), gentamicin (10 lg), kanamycin (30 lg), natilmicin (30 lg), novobiocin (5 lg), polymyxin B (300U), streptomycin (10 lg), tetracycline (30 lg), tobramycin (10 lg) and vancomycin (30 lg). The only difference between the two strains was that strain YIM 77502T was sensitive to penicillin G (10 IU) but not strain YIM 77510T. The major differential characteristics between strains YIM 77502T, YIM 77510T and the related type strains A. echinospora BCRC 12547T and A. umbrina KCTC 9343T are shown in Table 1. The detailed physiological characteristics of strains YIM 77502T and YIM 77510T are given in the species description. Chemotaxonomy The cell wall of the strains YIM 77502T and YIM 77510T contained meso-diaminopimelic acid, while the whole-cell sugars were glucose, madurose, mannose and rhamnose. The polar lipid profile of strain YIM 77502T was found to consist of diphosphatidylglycerol, phosphatidylinositol mannoside, phosphatidylinositol, two unidentified phospholipids and an unidentified polar lipid, while strain YIM 77510T consisted of diphosphatidylglycerol, phosphatidylinositol mannoside and phosphatidylinositol. The respiratory quinone for both strains were MK-9(H6) and
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MK-9(H8). The major fatty acids ([10 %) of strain YIM 77502T were C17:0, iso-C16:0, C17:0 10-methyl, iso-C18:0, while those of strain YIM 77510T were isoC16:0, C17:0 10-methyl, iso-C18:0, which is consistent with the profiles described for other members of the genus Actinomadura, though their proportions were different (Table 2). The G?C contents of strains YIM 77502T and YIM 77510T were determined to be 71.3 and 70.2 mol%, respectively. Phylogenetic analysis and DNA–DNA relatedness The almost complete 16S rRNA gene sequences of strains YIM 77502T (1540 bp) and YIM 77510T (1545 bp) were obtained. The GenBank accession numbers are AB859253 and AB859254, respectively. Sequence analysis of the almost complete 16S rRNA gene sequences of the two strains using the EzTaxon-e server showed that they had highest similarities to members of the genus Actinomadura, and are closely related to A. echinospora BCRC 12547T (97.6 and 97.3 % similarity) and A. umbrina KCTC 9343T (97.4 and 98.0 %). The two strains had 98.4 % similarity between them. The neighbour-joining phylogenetic tree based on 16S rRNA sequences showed the two strains form a clade with A. echinospora BCRC 12547T and A. umbrina KCTC 9343T, and were well separated from other members of the genus Actinomadura (Fig. 1). The stabilities of trees were further confirmed by maximum-likelihood and maximumparsimony methods (Figs. S3 and S4). The levels of DNA–DNA relatedness of strain YIM 77502T with A. echinospora BCRC 12547T and A. umbrina KCTC 9343T were 56.1 ± 2.5 and 44.9 ± 1.9 %, respectively, while the DNA–DNA hybridization values for strain YIM 77510T with respect to A. echinospora BCRC 12547T and A. umbrina KCTC 9343T were 10.2 ± 2.4 and 28.1 ± 3.4 %, respectively. The two strains YIM 77502T and YIM 77510T also had a DNA–DNA relatedness value of 36.9 ± 3.8 %, thereby indicating that all the DNA–DNA hybridization values were less than the cut-off point (70 %) for the delineation of genomic species (Stackebrandt and Goebel 1994). Therefore, on the basis of phylogenetic analysis, phenotypic and chemotaxonomic characteristics (Tables 1, 2; Fig. 1, Figs S1–S4), strains YIM 77502T and YIM 77510T should be affiliated to the genus Actinomadura. Strains YIM 77502T and YIM
Antonie van Leeuwenhoek Table 1 Different characteristics among strains YIM 77502T, YIM 77510T, A. echinospora BCRC 12547T and A. umbrina KCTC 9343T
Characteristics
YIM 77502T
YIM 77510T
BCRC 12547T
KCTC 9343T
Optimum temperature
45
45
35
30
NaCl tolerance(%)
3
3
2.5
2.5
Milk peptonization
?
?
-
-
Milk coagulation
?
-
-
-
Nitrate reduction
?
?
?
-
Tween 40
?
?
-
?
Starch Gelatin
? -
?
? ?
-
Cellulose
?
?
-
-
D-galactose
?
?
-
-
D-mannose
-
-
?
?
D-sorbitol
-
?
-
-
Glucose
?
?
-
?
Inositol
?
-
-
?
L-rhamnose
?
-
?
?
Mannitol
-
?
-
-
Xylitol
?
-
-
?
Arginine
?
-
?
-
Aspartic acid
?
-
?
-
DL-methionine
-
? -
? ?
-
Degradation of
Carbon source utilization
Nitrogen source utilization
Glycine
All the data were obtained from this study ?, positive, - negative
Hypoxanthine
?
-
-
-
L-histidine
-
-
?
-
L-threonine
?
?
?
-
L-tyrosine
?
?
?
-
L-valine
?
?
?
-
Lysine
?
-
?
-
Phenylalanine
-
-
?
-
Proline
?
?
?
-
Tryptophan
?
-
?
?
Xanthine
?
-
-
-
?
-
?
?
Sensitive to PenicillinG
77510T could be distinguished from the type strains A. echinospora BCRC 12547T and A. umbrina KCTC 9343T by several properties, such as temperature range and NaCl tolerance for growth, hydrolysis of cellulose, gelatin, starch, and Tween 40, utilization of carbon sources and nitrogen sources, and the proportions of some fatty acids. In addition, analyses of 16S rRNA gene sequences, DNA–DNA relatedness
values and their different properties, especially enzyme activities, the antibiotic sensitivity and utilization of sole-carbon sources and nitrogen sources, indicate that strains YIM 77502T and YIM 77510T represent two novel species within the genus Actinomadura, for which the names Actinomadura amylolytica sp. nov. and Actinomadura cellulosilytica sp. nov. are proposed, respectively.
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Antonie van Leeuwenhoek Table 2 Cellular fatty acid profiles (% of total) for strains YIM 77502T, YIM 77510T, Actinomadura echinospora BCRC 12547T and Actinomadura umbrina KCTC 9343T
Fatty acid (%)
YIM 77502T
YIM 77510T
BCRC 12547T
KCTC 9343T
Saturated fatty acids C16:0
2.8
3.9
4.8
4.7
C17:0
16.3
6.6
8.2
2.5
C18:0
2.2
5.0
3.7
6.3
C19:0
1.9
2.1
1.1
0.3
Branched fatty acids
All data were obtained in this study. Values are percentage of total fatty acid. ND not detected, Tr trace, mean values less than 0.5 %. Fatty acids that represent \0.5 % in all strains were omitted a
Summed in feature nine comprises 10-methyl C16:0; summed feature nine comprised iso-C17:1 x9c and/or 10-methyl C16:0
Iso-C16:0
37.5
26.3
42.7
46.1
C17:0 10-methyl Iso-C18:0
19.4 10.9
13.0 16.8
8.4 17.1
12.0 13.5
Iso-C14:0
1.0
0.5
1.9
0.9
Anteiso-C15:0
Tr
Tr
Tr
0.6
Iso-C15:0
Tr
0.8
ND
Tr
Anteiso-C17:0
1.6
4.1
0.7
0.6
Iso-C17:0
1.3
6.1
ND
Tr
Anteiso-C19:0
ND
1.0
ND
ND
Iso-C19:0
ND
0.5
ND
ND
C18:0 10-methyl
1.1
7.4
2.8
6.5
C19:0 10-methyl
ND
Tr
Tr
1.0
Unsaturated fatty acids C17:1x8c
2.0
1.5
3.7
Tr
C18:1x9c
Tr
0.8
1.4
0.5
Iso G-C16:1
ND
0.6
Tr
Tr
Tr
1.0
Tr
1.3
Sum In Feature 9a
Description of Actinomadura amylolytica sp. nov Actinomadura amylolytica (a.my.lo.ly0 ti.ca. Gr. n. amulon, starch; Gr. adj. lutikos, dissolving; N. L. fem. adj. amylolytica starch dissolving). Aerobic, Gram-staining positive; forms extensively branched, non-fragmenting, orange-yellow or yellowwhite substrate mycelia; bear white aerial mycelia on all the tested media. Aerial mycelia produce spiral spore chains with rugose surface. Diffusible pigments are produced on Gause’s synthetic agar. Growth occurs at 25–55 °C and pH 6.0–9.0 with optimal growth at 45 °C and pH 7.0. Can tolerate 0–3 % (w/v) NaCl. Positive for catalase, milk peptonization and coagulation, nitrate reduction, urease and cellulose, starch and Tween 40 hydrolysis tests, but negative for oxidase, H2S production and hydrolysis of gelatin and Tweens 20, 60 and 80. Utilizes D-fructose, D-galactose, glucose, inositol, L-rhamnose and xylitol as the sole carbon and energy sources, but not arabinose,
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cellose, a-lactose, maltose, mannitol, D-mannose, raffinose, sodium malate, D-sorbitol, succinic acid, sucrose, trehalose and xylose as the sole carbon sources. Alanine, arginine, aspartic acid, hypoxanthine, lysine, proline, serine, L-threonine, tryptophan, L-tyrosine, L-valine and xanthine can be used as the sole nitrogen sources, but not glutamic acid, glycine, L-histidine, DL-methionine or phenylalanine. The cell wall contains meso-DAP, whole-cell sugars contain glucose, madurose, mannose and rhamnose. The polar lipid profile consists of diphosphatidylglycerol, phosphatidylinositol mannoside, phosphatidylinositol, two unidentified phospholipids and an unidentified polar lipid. MK-9(H6) and MK-9(H8) are the predominant menaquinones. The major fatty acids ([10 %) are C17:0, iso-C16:0, C17:0 10-methyl, iso-C18:0. The G?C content of the type strain is 71.3 mol%. The type strain YIM 77502T (=DSM 45822T = CCTCC AA 2012024T) was isolated from the geothermally heated soil located in Tengchong
Antonie van Leeuwenhoek Fig. 1 Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences of strains YIM 77502T, YIM 77510T and their closest relatives. Bootstrap values (expressed as percentages of 1000 replications) above 50 % are shown at the branch points. Asterisks denote nodes that were also recovered using the maximum-parsimony and maximum-likelihood methods. The sequence of Streptomyces glauciniger FXJ14T was used as the outgroup. Bar 0.01, represents substitutions per nucleotide position
81* Actinomadura verrucosospora NBRC 14100T/U49011 66* Actinomadura coerulea IFO 14679T/U49002 97* Actinomadura citrea IFO 14678T/U49001 74* Actinomadura mexicana A290T/AF277195 Actinomadura xylanilytica BK147T/FR692101 Actinomadura bangladeshensis 3-46-b3T/AB331652 99* Actinomadura chokoriensis 3-45-a/11T/AB331730 93* Actinomadura geliboluensis A8036T/HQ157187 76* Actinomadura meyerae A288T/AY273787
*
Actinomadura yumaensis JCM 3369T/AF163122 Actinomadura chibensis IFM 10266T/AB264086
* * 62
Actinomadura livida JCM 3387T/AF163116 Actinomadura vinacea JCM 3325T/AF134070 99* Actinomadura viridis IFO 15238T/D85467
99* 56*
60* 85*
*
60 80
Actinomadura sputiIMMIB L-889T/FM957483 Actinomadura hallensis H647-1T/DQ076484 Actinomadura miaoliensis BC 44T-5T/EF116925 100* Actinomadura keratinilytica WCC-2265T/EU637009 Actinomadura atramentaria IFO 14695T/U49000 Actinomadura flavalba YIM 61435T/FJ157185 Actinomadura echinospora IFO 14042T/U49004 Actinomadura umbrina JCM 6837T/AF163121 Actinomadura cellulosilytica YIM 77510T/AB859254 Actinomadura amylolytica YIM 77502T/AB859253 Thermomonospora curvata DSM 43183T/CP001738 Streptomyces glauciniger FXJ14T/AY314782
0.01
county, Yunnan province, south-west China. The GenBank accession number for the 16S rRNA gene sequence of strain YIM 77502T is AB859253. Description of Actinomadura cellulosilytica sp. nov Actinomadura cellulosilytica (cel.lu.lo.si.ly’ ti.ca. N.L. n. cellulosum cellulose; N.L. adj. lyticus dissolving; N.L. fem. adj. cellulosilytica cellulose-dissolving). Aerobic, Gram-staining positive. Forms extensively branched, non-fragmenting, orange–yellow, yellowish–brown, yellow or yellow–white substrate mycelia; aerial mycelia differentiate into chains of spores with rugose surface. Diffusible pigments are not produced on any of the tested media. Growth occurs at 25–55 °C and pH 7.0–8.0 with optimal growth at 45 °C and at pH 7.0. Can tolerate up to 3 % (w/v) NaCl. Cellulose, gelatin, Tween 40 and urea are hydrolysed, but not Tweens (20, 60 and 80). Positive for catalase, milk peptonization and nitrate reduction but negative for milk coagulation, oxidase, H2S production and starch
hydrolysis tests. Utilizes D-fructose, D-galactose, glucose, mannitol and D-sorbitol as the sole carbon and energy sources, but not arabinose, cellulose, inositol, a-lactose, maltose, D-mannose, raffinose, L-rhamnose, sodium malate, succinic acid, sucrose, trehalose, xylitol and xylose as the sole carbon sources. Alanine, DL-methionine, proline, serine, L-threonine, L-tyrosine and L-valine can be used as the sole nitrogen sources, but not arginine, aspartic acid, glutamic acid, glycine, L-histidine, hypoxanthine, lysine, phenylalanine, tryptophan and xanthine. The cell wall contains mesoDAP, whole-cell sugars contain glucose, madurose, mannose and rhamnose. The polar lipids profile consists of diphosphatidylglycerol, phosphatidylinositol mannoside and phosphatidylinositol. The respiratory quinones are MK-9(H6) and MK-9(H8). The major fatty acids ([10 %) are iso-C16:0, C17:0 10-methy and iso-C18:0, The G?C content of the type strain is 70.2 mol%. The type strain YIM 77510T (=DSM 45823T = CCTCC AA 2012023T) was isolated from the geothermally heated soil located in Tengchong
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Antonie van Leeuwenhoek
county, Yunnan province, south-west China. The GenBank accession number for the 16S rRNA gene sequence of strain YIM 77510T is AB859254. Acknowledgments We are grateful to Dr. Jung-Sook Lee (KCTC) and Dr. Min Tseng (BCRC) for their kind providing reference type strains, and Prof. Aharon Oren (The Hebrew University of Jerusalem, Israel) for his kind help with the Latin etymology for the new species and Dr. Nimaichand Salam (Manipur University, India) for his help to further polishing this manuscript. This research was supported by Key Project of International Cooperation of Ministry of Science & Technology (MOST) (No. 2013DFA31980), Natural Science Foundation of China (No. 31470139) and Yunnan Provincial Natural Science Foundation (2013FA004). W-J Li was also supported by Guangdong Province Higher Vocational Colleges & Schools Pearl River Scholar Funded Scheme (2014).
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