Antonie van Leeuwenhoek (2014) 105:755–761 DOI 10.1007/s10482-014-0131-6
ORIGINAL PAPER
Zhizhongheella caldifontis gen. nov., sp. nov., a novel member of the family Comamonadaceae Lei Dong • Hong Ming • Lan Liu • En-Min Zhou Yi-Rui Yin • Yan-Yan Duan • Guo-Xing Nie • Hui-Geng Feng • Wen-Jun Li
•
Received: 10 December 2013 / Accepted: 29 January 2014 / Published online: 12 February 2014 Ó Springer International Publishing Switzerland 2014
Abstract An alkalitolerant, thermotolerant, strictly aerobic and Gram-staining negative bacterial strain, designated YIM 78140T, was isolated from a water sample in Hehua hot spring, Tengchong, Yunnan province, south-west China. The colonies were light brown, convex and circular. Phylogenetic analysis of the 16S rRNA gene sequence of strain YIM 78140T indicated that it was clustered with members of b-Proteobacteria (with the similarity from 96.9 to 93.6 %). Good growth occurred at 40–50 °C, pH
Lei Dong and Hong Ming contributed equally to this work.
Electronic supplementary material The online version of this article (doi:10.1007/s10482-014-0131-6) contains supplementary material, which is available to authorized users. L. Dong H. Ming L. Liu E.-M. Zhou Y.-R. Yin W.-J. Li (&) Key Laboratory of Microbial Diversity in Southwest China, Ministry of Education, and Laboratory for Conservation and Utilization of Bio-resources, Yunnan Institute of Microbiology, Yunnan University, Kunming 650091, People’s Republic of China e-mail: [email protected]; [email protected] H. Ming H.-G. Feng College of Life Sciences and Technology, Xinxiang Medical University, Xinxiang 453003, People’s Republic of China Y.-Y. Duan G.-X. Nie College of Fisheries, Henan Normal University, Xinxiang 453007, People’s Republic of China
8.0–9.0 and in the presence of 0–3 % (w/v) NaCl. The predominant ubiquinones were Q-8 and Q-9. The major fatty acids were C16:0, C17:0 cyclo, C18:1 x7c and summed feature 3. The G?C content of genomic DNA was 70.8 mol%. The results of physiological and biochemical characteristics, phylogenetic analysis allowed the phenotypic and genotypic differentiation of strain YIM 78140T from its closest phylogenetic neighbours. Therefore, the strain YIM 78140T represents a novel genus of the family Comamonadaceae, for which the name Zhizhongheella caldifontis gen. nov., sp. nov. is proposed. The type strain is YIM 78140T (= BCRC 80649T = KCTC 32557T). Keywords Family Comamonadaceae Zhizhongheella gen. nov. Zhizhongheella caldifontis sp. nov. Hehua hot spring Polyphasic taxonomy
Introduction The family Comamonadaceae was first described by (Willems et al. 1991). The members are Gram-staining negative and motile by means of either one polar flagellum. At the time of writing, 34 genera have been validly described (http://www.bacterio.net/). Strains of the family Comamonadaceae were obtained from soil, hot spring water, freshwater, waste water, activated sludge, pond water, salt mine and so on (Dubinina and Grabovich 1984; Takeda et al. 2002;
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Grabovich et al. 2006; Heylen et al. 2008; Hiraishi et al. 1995; Spring et al. 2005; Yu et al. 2011; Zhang et al. 2012), which indicated that this evolutionary cluster had a wide spectrum of habitat and varied metabolic pathways. Some strains were capable of degrading hydrocarbons (Rouvie`re and Chen 2003), accumulating phosphorous (Blackall et al. 2002; Lee et al. 2003), oxidizing ammonia (Juretschko et al. 2002; Purkhold et al. 2000) and performing denitrification (Ginige et al. 2004). Here we report the characterization of strain YIM 78140T, which was isolated from hot spring water in south-west China. During investigating thermophilic microbial resources at Hehua hot spring in Tengchong, Yunnan province, south-west China, we obtained strain YIM 78140T from the Hehua hot spring site. In the present study, polyphasic taxonomic study of strain YIM 78140T was carried out. Based on the morphological, physiological, chemotaxonomic results and phylogenetic analysis, strain YIM 78140T represents a novel genus of the family Comamonadaceae, for which the name Zhizhongheella caldifontis gen. nov., sp. nov. is proposed.
Materials and methods Strain YIM 78140T was isolated from a water sample (N 24.9728°, W 98.39706°). Sample collected from Hehua hot spring, Tengchong, Yunnan province, south-west China. After one week incubation on R2A medium (BD; Becton, Dickinson and Company) at 45 °C, the colony of strain YIM 78140T was picked and repeatedly re-streaked onto International Streptomyces Project agar medium (ISP) 2 (Shirling and Gottlieb 1966) at 45 °C, until purity was confirmed. The reference strains used (type strains) were obtained from RIKEN BioResource Center, Japan Collection of Microorganisms (JCM) for Caldimonas manganoxidans JCM 10698T and Bioresource Collection and Research Center (BCRC), Taiwan for Caldimonas taiwanensis BCRC 17405T. The purified strain YIM 78140T and reference type strains were routinely cultivated and stored as aqueous glycerol suspensions (20 %, v/v) at -80 °C. Since the reference type strains can also be cultivated on ISP 2 medium at 45 °C, biomasses for chemical and molecular studies were obtained for all three type strains by cultivation in
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shaken flasks (approximately 150 rpm.) by using ISP 2 broth medium at 45 °C for about 1 week. Morphological, cultural, physiological and biochemical characteristics Anaerobic growth was observed after incubation in an anaerobic chamber for 7 days at 45 °C on ISP 2 agar plate. Gram staining was carried out by using the standard gram reaction and was confirmed by using the KOH lysis test method (Cerny 1978). The morphological characteristics of strain YIM 78140T was observed by light microscopy (model BH 2; Olympus) and transmission electron microscopy (JEOL JEM-2100), after incubation on ISP 2 agar medium at 45 °C for 3 days. The procedure of preparing the sample for transmission electron microscopy was described by Ming et al. (2012). Growth temperature was tested at 4, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 and 60 °C on ISP 2 agar medium for 15 days. NaCl-tolerance tests were examined at different NaCl concentrations (0, 0.5, 1, 1.5, 2, 3, 5, 7, 10, 12, and 15 % w/v) ISP 2 agar medium at 45 °C. The pH growth range was investigated between pH 4.0 and 11.0 at intervals of 1 pH unit, by using the buffer system described by Yu et al. (2013), for growth was tested at 45 °C for 15 days by culturing the strain on ISP 2 broth medium. Carbon source utilization was investigated by the methods described by Shirling and Gottlieb (1966); Locci (1989). Biolog GN3 microplates were also used for characterization of singlecarbon-source assimilation and chemical sensitivity assays, according to the manufacturer’s instructions; plates were incubated at 45 °C and read after 48 h. Nitrogen source utilization was determined according to Williams et al. (1989). Catalase activity was detected by the production of bubbles after the addition of a drop of 3 % (v/v) H2O2. Oxidase activity was determined by the oxidation of tetramethyl-pphenylenediamine. Tests for hydrolysis of cellulose, gelatin, starch and Tweens 20, 40, 60 and 80, milk coagulation and peptonization, utilization of urea and nitrate reduction, were performed as described by Gonzalez et al. (1978). Enzyme activity and other biochemical characteristics were determined by using the API ZYM, API 20 NE and API 50 CH systems (bioMe´rieux, France) according to the manufacturer’s instructions.
Antonie van Leeuwenhoek (2014) 105:755–761
757
Table 1 Differential phenotypic characteristics of strain YIM 78140T and phylogenetically related taxa in the family Comamonadaceae Characteristic
1
2
3
4
5
Colony colour
Light brown
Creamy-grey
Light yellow
White
Cream or white
Oxidase
?
?
-
?
w
Catalase
?
?
w
?
-
Nitrate reduction
?
?
?
-
?
Glucose fermentation
?
-
-
-
-
Acetate
?
-
?
?
?
Adipate
?
-
?
-
?
Citrate
?
?
-
?
-
D-Arabinose
?
?
-
?
-
D-Fructose
-
?
?
-
-
D-Trehalose
-
-
?
-
-
D-Mannitol
-
?
?
-
-
Galactose
?
?
-
-
-
Malate
? ?
? ?
? -
? -
-
Maltose
?
?
?
-
?
Phenylacetate
-
-
?
-
?
Sucrose
-
?
-
-
-
Optimum growth temperature (°C)
40–50
50
55
50
50
Optimum growth pH
8.0–9.0
7.0–8.0
7.0
7.0
7.0
Isolation source
Hot spring
Hot spring
Hot spring
Activated sludge
Hot spring
70.8
65.9
66.2
70.0
69.2
Assimilation of
L-Glutamic
acid
DNA G?C content (mol%) T
T
Taxa: 1, YIM 78140 (data from this study); 2, Caldimonas manganoxidans JCM 10698 (data from this study); 3, Caldimonas taiwanensis On1T (data from this study); 4, Schlegelella thermodepolymerans K14T (Elbanna et al. 2003); 5, Schlegelella aquatica wcf1T (Chou et al. 2006). ‘‘?’’ positive, ‘‘-’’ negative, w weak reaction All the strains are positive for C4 esterase, C8 esterase lipase, leucine arylamidase, naphthol-AS-BI-phosphohydrolase and assimilation of gluconate, L-lactic acid, pyruvate and scucinic acid. The following characteristics are negative for all the strains: a-chymotrypsin, a-galactosidase, b-galactosidase, b-glucuronidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase, b-fucosidase and assimilation of adonitol, erythritol, D-mannose, L-rhamnose, inositol and xylitol
Chemotaxonomy
Molecular analysis
Biomass for chemical analysis was obtained by cultivation in shaken flasks by using ISP 2 broth medium at 45 °C for about 3 days. Cellular fatty acid analysis was performed by using the Microbial Identification System (Sherlock Version 6.1; MIDI database: TSBA6). The quinones were extracted by using the method of Collins et al. (1977) and separated by HPLC (Kroppenstedt 1982). The G?C DNA content of strain YIM 78140T was determined by using the HPLC method of Mesbah et al. (1989).
Extraction of genomic DNA and PCR amplification of the 16S rRNA gene were performed from strain YIM 78140T as described previously (Li et al. 2007). The resulting 16S RNA gene sequence was compared with available 16S rRNA gene sequences of cultured species from GenBank via the BLAST program and the EzTaxon-e server (http://eztaxon-e.ezbiocloud. net/; Kim et al. 2012). Multiple alignments with sequences of the most closely related taxa and calculations of levels of sequence similarity were carried out by using CLUSTAL_X (Thompson et al. 1997).
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Phylogenetic analyses were performed by using three tree-making algorithms via the software packages MEGA version 5.0 (Tamura et al. 2011) with three algorithms, the neighbour-joining (Saitou and Nei 1987), maximum-likelihood (Felsenstein 1981) and maximum-parsimony (Fitch 1971) methods. Kimura’s two parameter model was used to calculate Evolutionary distance matrices of the neighbour-joining method (Kimura 1980). Bootstrap analysis (1,000 resamplings) was used to evaluate the topology of phylogenetic trees (Felsenstein 1985).
Results and discussion The cells of strain YIM 78140T were aerobic, Gramstaining negative. Transmission electron micrograph of strain YIM 78140T showed that the cells were shortrod-shaped and with one single flagellum (Fig. S1). Growth occurred at 10–55 °C (optimum 40–50 °C), pH 6.0–10.0 (optimum 8.0–9.0) and in the presence of 0–3 % (w/v) NaCl. The strain was positive for oxidase, catalase, alkaline phosphatase, esterase, esterase lipase, leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, acid phosphatase and naphthol-AS-BI-phosphohydrolase, but negative for urease, amylase, cellulose. Tests of milk coagulation, milk peptonization, hydrolysis of Tweens 20, 40 are positive, but gelatin, pectin and H2S production are negative. The detailed physiological and biochemical characteristics are listed in the new species description, and the difference among strain YIM 78140T and its closely related phylogenetic neighbors are shown in Table 1. The analyses indicated that the major cellular fatty acid profile of strain YIM 78140T was composed of C16:0 (19.7 %), C17:0 cyclo (31.0 %), C18:1 x7c (11.3 %) and summed feature 3 (15.5 %). Strain YIM 78140T could be distinguished easily from the Schlegelella members of Schlegelella thermodepolymerans K14T; Schlegelella aquatica wcf1T; Caldimonas members of Caldimonas manganoxidans JCM 10698T; Caldimonas taiwanensis On1T based on the presence/absence and amount of C14:0, C15:1 x6c, C16:0 C17:0 cyclo and C18:1 x7c. The comparison of cellular fatty acid compositions of the novel strain YIM 78140T and related species of the family Comamonadaceae were listed in Table 2. The quinine system was composed of Q-8 and Q-9. The G?C
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content of the DNA of strain YIM 78140T was 70.8 mol%, which was slightly higher than those values for the other members of the family Comamonadaceae (65.9–70.0 mol%). The chemotaxonomic characteristics of strain YIM 78140T, such as ubiquinones and major fatty acids were consistent with its assignment to the family Comamonadaceae. The almost complete 16S rRNA gene sequence of strain YIM 78140T (1,548 bp) was determined and phylogenetic analysis indicated that strain YIM 78140T was related to members of the family Comamonadaceae in the class b-Proteobacteria and exhibited highest 16S rRNA gene sequence similarity to Caldimonas members of C. manganoxidans JCM 10698T (96.8 %); C. taiwanensis On1T (96.8 %); Schlegelella members of S. thermodepolymerans K14T (96.2 %); S. aquatica wcf1T (96.3 %). A total of 32 nearly full-length 16S rRNA gene sequences from reference strains were aligned with that of strain Table 2 Comparison of cellular fatty acid compositions (%) of strain YIM 78140T and related taxa of the family Comamonadaceae Fatty acids
1
2
3 ND
C9:0 3-OH
–
1.1
C10:0
–
–
4
5
–
–
1.3
1.2 5.1
C10:0 3-OH
4.5
11.1
6.3
3.3
C12:0
2.9
7.5
4.3
1.6
2.3
C12:0 3-OH
ND
–
ND
2.0
ND
C13:0 C14:0
– 3.2
1.2 5.7
– 2.9
ND ND
ND ND ND
C15:1 x6c
2.3
2.3
–
ND
C16:0
19.7
20.5
30.4
43.1
48.7
C16:1 x5c
2.6
1.5
–
ND
ND
C17:0
1.9
4.1
–
ND
ND 16.5
C17:0 cyclo
31.0
16.8
1.9
32.6
C18:0
–
–
2.1
–
1.0
C18:1 x7c
11.3
12.0
20.0
4.4
4.3
15.5
13.0
31.3
–
19.5
Summed feature 3a T
Taxa: 1, YIM 78140 (data from this study); 2, Caldimonas manganoxidans JCM 10698T (data from this study); 3, Caldimonas taiwanensis On1T (data from this study); 4, Schlegelella thermodepolymerans K14T; 5, Schlegelella aquatica wcf1T (data from previous study). ‘‘–’’ cellular fatty acid values of less than 1 %, ND not detected a
Summed features are groups of two or three fatty acids that cannot be separated by GLC with the MIDI system. Summed feature 3 comprises C16:1 x7c and/or C16:1 x6c. Cells were harvested after growth on ISP 2 medium at 45 °C for 3 days
Antonie van Leeuwenhoek (2014) 105:755–761
759 Leptothrix mobilis Feox-1T (X97071)
63 77 95*
Leptothrix cholodnii CCM 1827 (X97070) Leptothrix discophora SS-1T (L33975) Aquincola tertiaricarbonis L10T (DQ656489) Azohydromonas lata IAM 12599T (D88007) Azohydromonas australica IAM 12664T (AB188124) Polyangium brachysporum K481-B101T (AM410613) Piscinibacter aquaticus IMCC 1728T (DQ664244)
51
Rivibacter subsaxonicus BF49T (AM774413)
99* 68*
Methylibium petroleiphilum PM1T (CP000555)
54
Rhizobacter dauci H6T (AB297965)
96* 100*
Rhizobacter fulvus Gsoil 322T (AB245356)
Schlegelella aquatica wcf1T (DQ417336)
98*
Schlegelella thermodepolymerans K14T (AY152824) Zhizhongheella caldifontis YIM 78140T (KF771277 )
62*
Caldimonas manganoxidans JCM 10698T (AB008801)
63* 100*
100*
90
Caldimonas taiwanensis On1T (AY845052) 100 Hydrogenophaga spp. Tepidimonas spp. Burkholderia cepacia Ballard 717T (U96927)
0.01
Fig. 1 Neighbour-joining phylogenetic tree based on 16S rRNA gene sequence (1,548 bp) of strain YIM 78140T and the other members of b-Proteobacteria. Bootstrap values (expressed as percentages of 1,000 replications) of above 50 % are shown at
the branch points. Burkholderia cepacia Ballard 717T was used as an outgroup. Asterisks denote nodes that were also recovered using the maximum-parsimony and maximum-likelihood methods. Bar 0.01 substitutions per nucleotide position
YIM 78140T for phylogenetic analysis. It is apparent from the neighbour-joining phylogenetic tree (Fig. 1) that strain YIM 78140T formed a distinct clade with members of genera Caldimonas and Schlegelella, of which was supported by a high bootstrap value of 100 %. The same affiliation among strain YIM 78140T and its closest neighbors C. manganoxidans JCM 10698T, C. taiwanensis On1T, S. thermodepolymerans K14T and S. aquatica wcf1T was also supported by maximum parsimony and maximum-likelihood algorithms with bootstrap values of 100.0 and 99.0 %, respectively (Supplementary Figs. S2, S3). Therefore, strain YIM 78140T may be considered as a different genomic genus in the family Comamonadaceae. Besides the phylogenetic analysis based on 16S rRNA gene sequences, strain YIM 78140T could also be differentiated readily from C. manganoxidans JCM 10698T, C. taiwanensis On1T, S. thermodepolymerans K14T and S. aquatica wcf1T based on many phenotypic and chemotaxonomic characteristics shown in Tables 1 and 2. Particularly for the new isolate
78140T, it has the ability to grow well at pH 9.0 and positive for glucose fermentation, and with relatively higher DNA G?C content. Therefore, we propose that strain YIM 78140T should be considered to represent a novel species within a new genus, for which the name Zhizhongheella caldifontis gen. nov., sp. nov. is proposed. Description of Zhizhongheella gen. nov. Zhizhongheella [Zhi.zhong.he.el’la. N.L. fem. dim. N. Zhizhongheella named after Zhi-Zhong He (1939–2012), a Chinese microbiologist who devoted himself to the biology of thermophiles]. Cells are aerobic, thermotolerant, short-rod-shaped, and Gram-stain negative. Growth occurs at 10–55 °C and pH 6.0–10.0, with optimum growth at 40–50 °C and pH 8.0–9.0, respectively. The predominant ubiquinones are Q-8 and Q-9. Major fatty acids ([10 %) are C16:0, C17:0 cyclo, C18:1 x7c and summed feature 3 (C16:1 x7c and/or C16:1 x6c). The G?C content of
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genomic DNA of the type strain of the type species is 70.8 mol%. The type species is Zhizhongheella caldifontis. Description of Zhizhongheella caldifontis sp. nov. Zhizhongheella caldifontis (cal.di’fon’tis. L. adj. caldus, hot; L. n. fons fontis, a spring; N.L. gen. N. caldifontis, of a hot spring). In addition to the characteristics given in the genus description above, exhibits the following characteristics. Good growth is observed on ISP 2 and modified T5 media, colonies on ISP 2 medium are circular, convex and light brown. Growth occurs in the presence of 0–3 % (w/v) NaCl. Positive for oxidase, catalase, alkaline phosphatase, esterase, esterase lipase, leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, acid phosphatase and naphthol-AS-BI-phosphohydrolase, but negative for urease, amylase, cellulose. Tests for milk coagulation, milk peptonization, hydrolysis of Tweens 20, 40 are positive, but gelatin, pectin and H2S production are negative. Acetate, adipate, bromo-succinic acid, citric acid, dextrin, D-cellobiose, D-turanose, glycerol, Dglucose-6-PO4, D-fructose-6-PO4, D-xylose, D-arabinose, D-glucose, D-gluconic acid, D-saccharic acid, D-lactic acid methyl ester, lactose, L-lactic acid, galactose, gluconate, maltose, malate, methyl pyruvate, mycose, p-hydroxy-phtnylacetic acid, raffinose, sorbose and a-keto-glutaric acid can be utilized as sole carbon sources, but adonitol, capric acid, dulcitol, D-trehalose, D-mannose, D-galactose, D-fructose, D-fucose, D-glucuronic acid, inositol, L-rhamnose, L-arabinose, L-xylose, sorbitol, mannitol, mucic acid, phenylacetate, sucrose and xylitol are not utilized. Glucuronamide, L-glutamic acid, L-pyroglutamic acid, N-acetyl-b-D-mannosamine are utilized as solo nitrogen sources, but not L-alanine, L-arginine, L-aspartic acid, L-histidine and L-serine. Acid is produced from D-arabinose, fructose, glucose, lactose, mycose, sorbose and raffinose. The type strain, YIM 78140T (= BCRC 80649T = KCTC 32557T) was isolated from a hot spring water sample (N 24.9728°, W 98.39706°) from Hehua hot spring, Tengchong, Yunnan province, south-west China. The 16S rRNA gene sequence of strain YIM 78140T has been deposited in GenBank under the accession number KF771277.
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Antonie van Leeuwenhoek (2014) 105:755–761 Acknowledgments The authors are grateful to Prof. Dr. Takuji Kudo (JCM) and Mrs. Min Tseng (BCRC) for their kind providing reference type strains. This research was supported by the Key Project of International Cooperation of Ministry of Science & Technology (MOST) (No. 2013DFA31980), National Science Foundation grant (OISE-0968421) and Key Project of Yunnan Provincial Natural Science Foundation (2013FA004).
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761 species of ammonia oxidizers based on comparative 16S rRNA and amoA sequence analysis: implications for molecular diversity surveys. Appl Environ Microbiol 66:5368–5382 Rouvie`re PE, Chen MW (2003) Isolation of Brachymonas petroleovorans CHX, a novel cyclohexane-degrading bproteobacterium. FEMS Microbiol Lett 227:101–106 Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425 Shirling EB, Gottlieb D (1966) Methods for characterization of Streptomyces species. Int J Syst Bacteriol 16:313–340 Spring S, Wagner M, Schumann P, Ka¨mpfer P (2005) Malikia granosa gen. nov., sp. nov., a novel polyhydroxyalkanoateand polyphosphate-accumulating bacterium isolated from activated sludge, and reclassification of Pseudomonas spinosa as Malikia spinosa comb. nov. Int J Syst Evol Microbiol 55:621–629 Takeda M, Kamagata Y, Ghiorse WC, Hanada S, Koizumi J (2002) Caldimonas manganoxidans gen. nov., sp. nov., a poly(3-hydroxybutyrate)-degrading, manganese-oxidizing thermophile. Int J Syst Evol Microbiol 52:895–900 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 Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882 Williams ST, Goodfellow M, Alderson G (1989) Genus Streptomyces Waksman and Henrici 1943, 339AL. In: Williams ST, Sharpe ME, Holt JG (eds) Bergey’s manual of systematic bacteriology, vol. 4. Williams & Wilkins, Baltimore, pp 2463–2468 Willems A, Pot B, Falsen E, Vandamme P, Gillis M, Kersters K, De Ley J (1991) Polyphasic taxonomic study of the emended genus Comamonas: relationship to Aquaspirillum aquaticum, E. Falsen group 10, and other clinical isolates. Int J Syst Bacteriol 41:427–444 Yu XY, Li YF, Zheng JW, Li Y, Li L, He J, Li SP (2011) Comamonas zonglianii sp. nov., isolated from phenolcontaminated soil. Int J Syst Evol Microbiol 61:255–258 Yu TT, Zhou EM, Yin YR, Yao JC, Ming H, Dong L, Li S, Nie GX & Li WJ (2013) Vulcaniibacterium tengchongense gen. nov., sp. nov. isolated from a geothermally heated soil sample, and reclassification of Lysobacter thermophilus Wei et al. 2012 as Vulcaniibacterium thermophilum comb. nov. Antonie van Leeuwenhoek: 1–8 Zhang WY, Fang MX, Zhang WW, Xiao C, Zhang XQ, Yu ZP, Zhu XF, Wu M (2012) Extensimonas vulgaris gen. nov., sp. nov., a member of the family Comamonadaceae. Int J Syst Evol Microbiol 63:2062–2068
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ORIGINAL PAPER
Zhizhongheella caldifontis gen. nov., sp. nov., a novel member of the family Comamonadaceae Lei Dong • Hong Ming • Lan Liu • En-Min Zhou Yi-Rui Yin • Yan-Yan Duan • Guo-Xing Nie • Hui-Geng Feng • Wen-Jun Li
•
Received: 10 December 2013 / Accepted: 29 January 2014 / Published online: 12 February 2014 Ó Springer International Publishing Switzerland 2014
Abstract An alkalitolerant, thermotolerant, strictly aerobic and Gram-staining negative bacterial strain, designated YIM 78140T, was isolated from a water sample in Hehua hot spring, Tengchong, Yunnan province, south-west China. The colonies were light brown, convex and circular. Phylogenetic analysis of the 16S rRNA gene sequence of strain YIM 78140T indicated that it was clustered with members of b-Proteobacteria (with the similarity from 96.9 to 93.6 %). Good growth occurred at 40–50 °C, pH
Lei Dong and Hong Ming contributed equally to this work.
Electronic supplementary material The online version of this article (doi:10.1007/s10482-014-0131-6) contains supplementary material, which is available to authorized users. L. Dong H. Ming L. Liu E.-M. Zhou Y.-R. Yin W.-J. Li (&) Key Laboratory of Microbial Diversity in Southwest China, Ministry of Education, and Laboratory for Conservation and Utilization of Bio-resources, Yunnan Institute of Microbiology, Yunnan University, Kunming 650091, People’s Republic of China e-mail: [email protected]; [email protected] H. Ming H.-G. Feng College of Life Sciences and Technology, Xinxiang Medical University, Xinxiang 453003, People’s Republic of China Y.-Y. Duan G.-X. Nie College of Fisheries, Henan Normal University, Xinxiang 453007, People’s Republic of China
8.0–9.0 and in the presence of 0–3 % (w/v) NaCl. The predominant ubiquinones were Q-8 and Q-9. The major fatty acids were C16:0, C17:0 cyclo, C18:1 x7c and summed feature 3. The G?C content of genomic DNA was 70.8 mol%. The results of physiological and biochemical characteristics, phylogenetic analysis allowed the phenotypic and genotypic differentiation of strain YIM 78140T from its closest phylogenetic neighbours. Therefore, the strain YIM 78140T represents a novel genus of the family Comamonadaceae, for which the name Zhizhongheella caldifontis gen. nov., sp. nov. is proposed. The type strain is YIM 78140T (= BCRC 80649T = KCTC 32557T). Keywords Family Comamonadaceae Zhizhongheella gen. nov. Zhizhongheella caldifontis sp. nov. Hehua hot spring Polyphasic taxonomy
Introduction The family Comamonadaceae was first described by (Willems et al. 1991). The members are Gram-staining negative and motile by means of either one polar flagellum. At the time of writing, 34 genera have been validly described (http://www.bacterio.net/). Strains of the family Comamonadaceae were obtained from soil, hot spring water, freshwater, waste water, activated sludge, pond water, salt mine and so on (Dubinina and Grabovich 1984; Takeda et al. 2002;
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Grabovich et al. 2006; Heylen et al. 2008; Hiraishi et al. 1995; Spring et al. 2005; Yu et al. 2011; Zhang et al. 2012), which indicated that this evolutionary cluster had a wide spectrum of habitat and varied metabolic pathways. Some strains were capable of degrading hydrocarbons (Rouvie`re and Chen 2003), accumulating phosphorous (Blackall et al. 2002; Lee et al. 2003), oxidizing ammonia (Juretschko et al. 2002; Purkhold et al. 2000) and performing denitrification (Ginige et al. 2004). Here we report the characterization of strain YIM 78140T, which was isolated from hot spring water in south-west China. During investigating thermophilic microbial resources at Hehua hot spring in Tengchong, Yunnan province, south-west China, we obtained strain YIM 78140T from the Hehua hot spring site. In the present study, polyphasic taxonomic study of strain YIM 78140T was carried out. Based on the morphological, physiological, chemotaxonomic results and phylogenetic analysis, strain YIM 78140T represents a novel genus of the family Comamonadaceae, for which the name Zhizhongheella caldifontis gen. nov., sp. nov. is proposed.
Materials and methods Strain YIM 78140T was isolated from a water sample (N 24.9728°, W 98.39706°). Sample collected from Hehua hot spring, Tengchong, Yunnan province, south-west China. After one week incubation on R2A medium (BD; Becton, Dickinson and Company) at 45 °C, the colony of strain YIM 78140T was picked and repeatedly re-streaked onto International Streptomyces Project agar medium (ISP) 2 (Shirling and Gottlieb 1966) at 45 °C, until purity was confirmed. The reference strains used (type strains) were obtained from RIKEN BioResource Center, Japan Collection of Microorganisms (JCM) for Caldimonas manganoxidans JCM 10698T and Bioresource Collection and Research Center (BCRC), Taiwan for Caldimonas taiwanensis BCRC 17405T. The purified strain YIM 78140T and reference type strains were routinely cultivated and stored as aqueous glycerol suspensions (20 %, v/v) at -80 °C. Since the reference type strains can also be cultivated on ISP 2 medium at 45 °C, biomasses for chemical and molecular studies were obtained for all three type strains by cultivation in
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shaken flasks (approximately 150 rpm.) by using ISP 2 broth medium at 45 °C for about 1 week. Morphological, cultural, physiological and biochemical characteristics Anaerobic growth was observed after incubation in an anaerobic chamber for 7 days at 45 °C on ISP 2 agar plate. Gram staining was carried out by using the standard gram reaction and was confirmed by using the KOH lysis test method (Cerny 1978). The morphological characteristics of strain YIM 78140T was observed by light microscopy (model BH 2; Olympus) and transmission electron microscopy (JEOL JEM-2100), after incubation on ISP 2 agar medium at 45 °C for 3 days. The procedure of preparing the sample for transmission electron microscopy was described by Ming et al. (2012). Growth temperature was tested at 4, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 and 60 °C on ISP 2 agar medium for 15 days. NaCl-tolerance tests were examined at different NaCl concentrations (0, 0.5, 1, 1.5, 2, 3, 5, 7, 10, 12, and 15 % w/v) ISP 2 agar medium at 45 °C. The pH growth range was investigated between pH 4.0 and 11.0 at intervals of 1 pH unit, by using the buffer system described by Yu et al. (2013), for growth was tested at 45 °C for 15 days by culturing the strain on ISP 2 broth medium. Carbon source utilization was investigated by the methods described by Shirling and Gottlieb (1966); Locci (1989). Biolog GN3 microplates were also used for characterization of singlecarbon-source assimilation and chemical sensitivity assays, according to the manufacturer’s instructions; plates were incubated at 45 °C and read after 48 h. Nitrogen source utilization was determined according to Williams et al. (1989). Catalase activity was detected by the production of bubbles after the addition of a drop of 3 % (v/v) H2O2. Oxidase activity was determined by the oxidation of tetramethyl-pphenylenediamine. Tests for hydrolysis of cellulose, gelatin, starch and Tweens 20, 40, 60 and 80, milk coagulation and peptonization, utilization of urea and nitrate reduction, were performed as described by Gonzalez et al. (1978). Enzyme activity and other biochemical characteristics were determined by using the API ZYM, API 20 NE and API 50 CH systems (bioMe´rieux, France) according to the manufacturer’s instructions.
Antonie van Leeuwenhoek (2014) 105:755–761
757
Table 1 Differential phenotypic characteristics of strain YIM 78140T and phylogenetically related taxa in the family Comamonadaceae Characteristic
1
2
3
4
5
Colony colour
Light brown
Creamy-grey
Light yellow
White
Cream or white
Oxidase
?
?
-
?
w
Catalase
?
?
w
?
-
Nitrate reduction
?
?
?
-
?
Glucose fermentation
?
-
-
-
-
Acetate
?
-
?
?
?
Adipate
?
-
?
-
?
Citrate
?
?
-
?
-
D-Arabinose
?
?
-
?
-
D-Fructose
-
?
?
-
-
D-Trehalose
-
-
?
-
-
D-Mannitol
-
?
?
-
-
Galactose
?
?
-
-
-
Malate
? ?
? ?
? -
? -
-
Maltose
?
?
?
-
?
Phenylacetate
-
-
?
-
?
Sucrose
-
?
-
-
-
Optimum growth temperature (°C)
40–50
50
55
50
50
Optimum growth pH
8.0–9.0
7.0–8.0
7.0
7.0
7.0
Isolation source
Hot spring
Hot spring
Hot spring
Activated sludge
Hot spring
70.8
65.9
66.2
70.0
69.2
Assimilation of
L-Glutamic
acid
DNA G?C content (mol%) T
T
Taxa: 1, YIM 78140 (data from this study); 2, Caldimonas manganoxidans JCM 10698 (data from this study); 3, Caldimonas taiwanensis On1T (data from this study); 4, Schlegelella thermodepolymerans K14T (Elbanna et al. 2003); 5, Schlegelella aquatica wcf1T (Chou et al. 2006). ‘‘?’’ positive, ‘‘-’’ negative, w weak reaction All the strains are positive for C4 esterase, C8 esterase lipase, leucine arylamidase, naphthol-AS-BI-phosphohydrolase and assimilation of gluconate, L-lactic acid, pyruvate and scucinic acid. The following characteristics are negative for all the strains: a-chymotrypsin, a-galactosidase, b-galactosidase, b-glucuronidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase, b-fucosidase and assimilation of adonitol, erythritol, D-mannose, L-rhamnose, inositol and xylitol
Chemotaxonomy
Molecular analysis
Biomass for chemical analysis was obtained by cultivation in shaken flasks by using ISP 2 broth medium at 45 °C for about 3 days. Cellular fatty acid analysis was performed by using the Microbial Identification System (Sherlock Version 6.1; MIDI database: TSBA6). The quinones were extracted by using the method of Collins et al. (1977) and separated by HPLC (Kroppenstedt 1982). The G?C DNA content of strain YIM 78140T was determined by using the HPLC method of Mesbah et al. (1989).
Extraction of genomic DNA and PCR amplification of the 16S rRNA gene were performed from strain YIM 78140T as described previously (Li et al. 2007). The resulting 16S RNA gene sequence was compared with available 16S rRNA gene sequences of cultured species from GenBank via the BLAST program and the EzTaxon-e server (http://eztaxon-e.ezbiocloud. net/; Kim et al. 2012). Multiple alignments with sequences of the most closely related taxa and calculations of levels of sequence similarity were carried out by using CLUSTAL_X (Thompson et al. 1997).
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Phylogenetic analyses were performed by using three tree-making algorithms via the software packages MEGA version 5.0 (Tamura et al. 2011) with three algorithms, the neighbour-joining (Saitou and Nei 1987), maximum-likelihood (Felsenstein 1981) and maximum-parsimony (Fitch 1971) methods. Kimura’s two parameter model was used to calculate Evolutionary distance matrices of the neighbour-joining method (Kimura 1980). Bootstrap analysis (1,000 resamplings) was used to evaluate the topology of phylogenetic trees (Felsenstein 1985).
Results and discussion The cells of strain YIM 78140T were aerobic, Gramstaining negative. Transmission electron micrograph of strain YIM 78140T showed that the cells were shortrod-shaped and with one single flagellum (Fig. S1). Growth occurred at 10–55 °C (optimum 40–50 °C), pH 6.0–10.0 (optimum 8.0–9.0) and in the presence of 0–3 % (w/v) NaCl. The strain was positive for oxidase, catalase, alkaline phosphatase, esterase, esterase lipase, leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, acid phosphatase and naphthol-AS-BI-phosphohydrolase, but negative for urease, amylase, cellulose. Tests of milk coagulation, milk peptonization, hydrolysis of Tweens 20, 40 are positive, but gelatin, pectin and H2S production are negative. The detailed physiological and biochemical characteristics are listed in the new species description, and the difference among strain YIM 78140T and its closely related phylogenetic neighbors are shown in Table 1. The analyses indicated that the major cellular fatty acid profile of strain YIM 78140T was composed of C16:0 (19.7 %), C17:0 cyclo (31.0 %), C18:1 x7c (11.3 %) and summed feature 3 (15.5 %). Strain YIM 78140T could be distinguished easily from the Schlegelella members of Schlegelella thermodepolymerans K14T; Schlegelella aquatica wcf1T; Caldimonas members of Caldimonas manganoxidans JCM 10698T; Caldimonas taiwanensis On1T based on the presence/absence and amount of C14:0, C15:1 x6c, C16:0 C17:0 cyclo and C18:1 x7c. The comparison of cellular fatty acid compositions of the novel strain YIM 78140T and related species of the family Comamonadaceae were listed in Table 2. The quinine system was composed of Q-8 and Q-9. The G?C
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content of the DNA of strain YIM 78140T was 70.8 mol%, which was slightly higher than those values for the other members of the family Comamonadaceae (65.9–70.0 mol%). The chemotaxonomic characteristics of strain YIM 78140T, such as ubiquinones and major fatty acids were consistent with its assignment to the family Comamonadaceae. The almost complete 16S rRNA gene sequence of strain YIM 78140T (1,548 bp) was determined and phylogenetic analysis indicated that strain YIM 78140T was related to members of the family Comamonadaceae in the class b-Proteobacteria and exhibited highest 16S rRNA gene sequence similarity to Caldimonas members of C. manganoxidans JCM 10698T (96.8 %); C. taiwanensis On1T (96.8 %); Schlegelella members of S. thermodepolymerans K14T (96.2 %); S. aquatica wcf1T (96.3 %). A total of 32 nearly full-length 16S rRNA gene sequences from reference strains were aligned with that of strain Table 2 Comparison of cellular fatty acid compositions (%) of strain YIM 78140T and related taxa of the family Comamonadaceae Fatty acids
1
2
3 ND
C9:0 3-OH
–
1.1
C10:0
–
–
4
5
–
–
1.3
1.2 5.1
C10:0 3-OH
4.5
11.1
6.3
3.3
C12:0
2.9
7.5
4.3
1.6
2.3
C12:0 3-OH
ND
–
ND
2.0
ND
C13:0 C14:0
– 3.2
1.2 5.7
– 2.9
ND ND
ND ND ND
C15:1 x6c
2.3
2.3
–
ND
C16:0
19.7
20.5
30.4
43.1
48.7
C16:1 x5c
2.6
1.5
–
ND
ND
C17:0
1.9
4.1
–
ND
ND 16.5
C17:0 cyclo
31.0
16.8
1.9
32.6
C18:0
–
–
2.1
–
1.0
C18:1 x7c
11.3
12.0
20.0
4.4
4.3
15.5
13.0
31.3
–
19.5
Summed feature 3a T
Taxa: 1, YIM 78140 (data from this study); 2, Caldimonas manganoxidans JCM 10698T (data from this study); 3, Caldimonas taiwanensis On1T (data from this study); 4, Schlegelella thermodepolymerans K14T; 5, Schlegelella aquatica wcf1T (data from previous study). ‘‘–’’ cellular fatty acid values of less than 1 %, ND not detected a
Summed features are groups of two or three fatty acids that cannot be separated by GLC with the MIDI system. Summed feature 3 comprises C16:1 x7c and/or C16:1 x6c. Cells were harvested after growth on ISP 2 medium at 45 °C for 3 days
Antonie van Leeuwenhoek (2014) 105:755–761
759 Leptothrix mobilis Feox-1T (X97071)
63 77 95*
Leptothrix cholodnii CCM 1827 (X97070) Leptothrix discophora SS-1T (L33975) Aquincola tertiaricarbonis L10T (DQ656489) Azohydromonas lata IAM 12599T (D88007) Azohydromonas australica IAM 12664T (AB188124) Polyangium brachysporum K481-B101T (AM410613) Piscinibacter aquaticus IMCC 1728T (DQ664244)
51
Rivibacter subsaxonicus BF49T (AM774413)
99* 68*
Methylibium petroleiphilum PM1T (CP000555)
54
Rhizobacter dauci H6T (AB297965)
96* 100*
Rhizobacter fulvus Gsoil 322T (AB245356)
Schlegelella aquatica wcf1T (DQ417336)
98*
Schlegelella thermodepolymerans K14T (AY152824) Zhizhongheella caldifontis YIM 78140T (KF771277 )
62*
Caldimonas manganoxidans JCM 10698T (AB008801)
63* 100*
100*
90
Caldimonas taiwanensis On1T (AY845052) 100 Hydrogenophaga spp. Tepidimonas spp. Burkholderia cepacia Ballard 717T (U96927)
0.01
Fig. 1 Neighbour-joining phylogenetic tree based on 16S rRNA gene sequence (1,548 bp) of strain YIM 78140T and the other members of b-Proteobacteria. Bootstrap values (expressed as percentages of 1,000 replications) of above 50 % are shown at
the branch points. Burkholderia cepacia Ballard 717T was used as an outgroup. Asterisks denote nodes that were also recovered using the maximum-parsimony and maximum-likelihood methods. Bar 0.01 substitutions per nucleotide position
YIM 78140T for phylogenetic analysis. It is apparent from the neighbour-joining phylogenetic tree (Fig. 1) that strain YIM 78140T formed a distinct clade with members of genera Caldimonas and Schlegelella, of which was supported by a high bootstrap value of 100 %. The same affiliation among strain YIM 78140T and its closest neighbors C. manganoxidans JCM 10698T, C. taiwanensis On1T, S. thermodepolymerans K14T and S. aquatica wcf1T was also supported by maximum parsimony and maximum-likelihood algorithms with bootstrap values of 100.0 and 99.0 %, respectively (Supplementary Figs. S2, S3). Therefore, strain YIM 78140T may be considered as a different genomic genus in the family Comamonadaceae. Besides the phylogenetic analysis based on 16S rRNA gene sequences, strain YIM 78140T could also be differentiated readily from C. manganoxidans JCM 10698T, C. taiwanensis On1T, S. thermodepolymerans K14T and S. aquatica wcf1T based on many phenotypic and chemotaxonomic characteristics shown in Tables 1 and 2. Particularly for the new isolate
78140T, it has the ability to grow well at pH 9.0 and positive for glucose fermentation, and with relatively higher DNA G?C content. Therefore, we propose that strain YIM 78140T should be considered to represent a novel species within a new genus, for which the name Zhizhongheella caldifontis gen. nov., sp. nov. is proposed. Description of Zhizhongheella gen. nov. Zhizhongheella [Zhi.zhong.he.el’la. N.L. fem. dim. N. Zhizhongheella named after Zhi-Zhong He (1939–2012), a Chinese microbiologist who devoted himself to the biology of thermophiles]. Cells are aerobic, thermotolerant, short-rod-shaped, and Gram-stain negative. Growth occurs at 10–55 °C and pH 6.0–10.0, with optimum growth at 40–50 °C and pH 8.0–9.0, respectively. The predominant ubiquinones are Q-8 and Q-9. Major fatty acids ([10 %) are C16:0, C17:0 cyclo, C18:1 x7c and summed feature 3 (C16:1 x7c and/or C16:1 x6c). The G?C content of
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genomic DNA of the type strain of the type species is 70.8 mol%. The type species is Zhizhongheella caldifontis. Description of Zhizhongheella caldifontis sp. nov. Zhizhongheella caldifontis (cal.di’fon’tis. L. adj. caldus, hot; L. n. fons fontis, a spring; N.L. gen. N. caldifontis, of a hot spring). In addition to the characteristics given in the genus description above, exhibits the following characteristics. Good growth is observed on ISP 2 and modified T5 media, colonies on ISP 2 medium are circular, convex and light brown. Growth occurs in the presence of 0–3 % (w/v) NaCl. Positive for oxidase, catalase, alkaline phosphatase, esterase, esterase lipase, leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, acid phosphatase and naphthol-AS-BI-phosphohydrolase, but negative for urease, amylase, cellulose. Tests for milk coagulation, milk peptonization, hydrolysis of Tweens 20, 40 are positive, but gelatin, pectin and H2S production are negative. Acetate, adipate, bromo-succinic acid, citric acid, dextrin, D-cellobiose, D-turanose, glycerol, Dglucose-6-PO4, D-fructose-6-PO4, D-xylose, D-arabinose, D-glucose, D-gluconic acid, D-saccharic acid, D-lactic acid methyl ester, lactose, L-lactic acid, galactose, gluconate, maltose, malate, methyl pyruvate, mycose, p-hydroxy-phtnylacetic acid, raffinose, sorbose and a-keto-glutaric acid can be utilized as sole carbon sources, but adonitol, capric acid, dulcitol, D-trehalose, D-mannose, D-galactose, D-fructose, D-fucose, D-glucuronic acid, inositol, L-rhamnose, L-arabinose, L-xylose, sorbitol, mannitol, mucic acid, phenylacetate, sucrose and xylitol are not utilized. Glucuronamide, L-glutamic acid, L-pyroglutamic acid, N-acetyl-b-D-mannosamine are utilized as solo nitrogen sources, but not L-alanine, L-arginine, L-aspartic acid, L-histidine and L-serine. Acid is produced from D-arabinose, fructose, glucose, lactose, mycose, sorbose and raffinose. The type strain, YIM 78140T (= BCRC 80649T = KCTC 32557T) was isolated from a hot spring water sample (N 24.9728°, W 98.39706°) from Hehua hot spring, Tengchong, Yunnan province, south-west China. The 16S rRNA gene sequence of strain YIM 78140T has been deposited in GenBank under the accession number KF771277.
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Antonie van Leeuwenhoek (2014) 105:755–761 Acknowledgments The authors are grateful to Prof. Dr. Takuji Kudo (JCM) and Mrs. Min Tseng (BCRC) for their kind providing reference type strains. This research was supported by the Key Project of International Cooperation of Ministry of Science & Technology (MOST) (No. 2013DFA31980), National Science Foundation grant (OISE-0968421) and Key Project of Yunnan Provincial Natural Science Foundation (2013FA004).
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