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

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 8.0–9.0 and in the presence of 0–3 % (w/v) NaCl. The aerobic and Gram-staining negative bacterial strain, predominant ubiquinones were Q-8 and Q-9. The T designated YIM 78140 , was isolated from a water major fatty acids were C16:0, C17:0 cyclo, C18:1 x7c and sample in Hehua hot spring, Tengchong, Yunnan summed feature 3. The G?C content of genomic DNA province, south-west China. The colonies were light was 70.8 mol%. The results of physiological and brown, convex and circular. Phylogenetic analysis of biochemical characteristics, phylogenetic analysis the 16S rRNA gene sequence of strain YIM 78140T allowed the phenotypic and genotypic differentiation indicated that it was clustered with members of of strain YIM 78140T from its closest phylogenetic b- (with the similarity from 96.9 to neighbours. Therefore, the strain YIM 78140T repre- 93.6 %). Good growth occurred at 40–50 °C, pH sents 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). Lei Dong and Hong Ming contributed equally to this work.

Electronic supplementary material The online version of Keywords Family Comamonadaceae Á this article (doi:10.1007/s10482-014-0131-6) contains supple- Zhizhongheella gen. nov. Á Zhizhongheella mentary material, which is available to authorized users. caldifontis sp. nov. Á Hehua hot spring Á L. Dong Á H. Ming Á L. Liu Á E.-M. Zhou Á Polyphasic Y.-R. Yin Á W.-J. Li (&) Key Laboratory of Microbial Diversity in Southwest China, Ministry of Education, and Laboratory for Introduction Conservation and Utilization of Bio-resources, Yunnan Institute of Microbiology, Yunnan University, Kunming 650091, People’s Republic of China The family Comamonadaceae was first described by e-mail: [email protected]; [email protected] (Willems et al. 1991). The members are Gram-staining negative and motile by means of either one polar H. Ming Á H.-G. Feng College of Life Sciences and Technology, Xinxiang flagellum. At the time of writing, 34 genera have been Medical University, Xinxiang 453003, People’s Republic validly described (http://www.bacterio.net/). Strains of China of the family Comamonadaceae were obtained from soil, hot spring water, freshwater, waste water, acti- Y.-Y. Duan Á G.-X. Nie College of Fisheries, Henan Normal University, vated sludge, pond water, salt mine and so on (Du- Xinxiang 453007, People’s Republic of China binina and Grabovich 1984; Takeda et al. 2002; 123 756 Antonie van Leeuwenhoek (2014) 105:755–761

Grabovich et al. 2006; Heylen et al. 2008; Hiraishi shaken flasks (approximately 150 rpm.) by using ISP 2 et al. 1995; Spring et al. 2005; Yu et al. 2011; Zhang broth medium at 45 °C for about 1 week. et al. 2012), which indicated that this evolutionary cluster had a wide spectrum of habitat and varied Morphological, cultural, physiological metabolic pathways. Some strains were capable of and biochemical characteristics degrading hydrocarbons (Rouvie`re and Chen 2003), accumulating phosphorous (Blackall et al. 2002; Lee Anaerobic growth was observed after incubation in an et al. 2003), oxidizing ammonia (Juretschko et al. anaerobic chamber for 7 days at 45 °C on ISP 2 agar 2002; Purkhold et al. 2000) and performing denitrifi- plate. Gram staining was carried out by using the cation (Ginige et al. 2004). Here we report the char- standard gram reaction and was confirmed by using acterization of strain YIM 78140T, which was isolated the KOH lysis test method (Cerny 1978). The from hot spring water in south-west China. morphological characteristics of strain YIM 78140T During investigating thermophilic microbial was observed by light microscopy (model BH 2; resources at Hehua hot spring in Tengchong, Yunnan Olympus) and transmission electron microscopy province, south-west China, we obtained strain YIM (JEOL JEM-2100), after incubation on ISP 2 agar 78140T from the Hehua hot spring site. In the present medium at 45 °C for 3 days. The procedure of study, polyphasic taxonomic study of strain YIM preparing the sample for transmission electron micros- 78140T was carried out. Based on the morphological, copy was described by Ming et al. (2012). Growth physiological, chemotaxonomic results and phyloge- temperature was tested at 4, 10, 15, 20, 25, 30, 35, 40, netic analysis, strain YIM 78140T represents a novel 45, 50, 55 and 60 °C on ISP 2 agar medium for genus of the family Comamonadaceae, for which the 15 days. NaCl-tolerance tests were examined at name Zhizhongheella caldifontis gen. nov., sp. nov. is different NaCl concentrations (0, 0.5, 1, 1.5, 2, 3, 5, proposed. 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 Materials and methods system described by Yu et al. (2013), for growth was tested at 45 °C for 15 days by culturing the strain on Strain YIM 78140T was isolated from a water sample ISP 2 broth medium. Carbon source utilization was (N 24.9728°, W 98.39706°). Sample collected from investigated by the methods described by Shirling and Hehua hot spring, Tengchong, Yunnan province, Gottlieb (1966); Locci (1989). Biolog GN3 micro- south-west China. After one week incubation on plates were also used for characterization of single- R2A medium (BD; Becton, Dickinson and Company) carbon-source assimilation and chemical sensitivity at 45 °C, the colony of strain YIM 78140T was picked assays, according to the manufacturer’s instructions; and repeatedly re-streaked onto International Strepto- plates were incubated at 45 °C and read after 48 h. myces Project agar medium (ISP) 2 (Shirling and Nitrogen source utilization was determined according Gottlieb 1966)at45°C, until purity was confirmed. to Williams et al. (1989). Catalase activity was The reference strains used (type strains) were obtained detected by the production of bubbles after the from RIKEN BioResource Center, Japan Collection of addition of a drop of 3 % (v/v) H2O2. Oxidase activity Microorganisms (JCM) for Caldimonas manganoxi- was determined by the oxidation of tetramethyl-p- dans JCM 10698T and Bioresource Collection and phenylenediamine. Tests for hydrolysis of cellulose, Research Center (BCRC), Taiwan for Caldimonas gelatin, starch and Tweens 20, 40, 60 and 80, milk taiwanensis BCRC 17405T. The purified strain YIM coagulation and peptonization, utilization of urea and 78140T and reference type strains were routinely nitrate reduction, were performed as described by cultivated and stored as aqueous glycerol suspensions Gonzalez et al. (1978). activity and other (20 %, v/v) at -80 °C. Since the reference type strains biochemical characteristics were determined by using can also be cultivated on ISP 2 medium at 45 °C, the API ZYM, API 20 NE and API 50 CH systems biomasses for chemical and molecular studies were (bioMe´rieux, France) according to the manufacturer’s obtained for all three type strains by cultivation in instructions.

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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 ?- - - - Assimilation of Acetate ?- ? ? ? Adipate ?- ? - ? Citrate ?? - ? - D-Arabinose ?? - ? - D-Fructose -? ? - - D-Trehalose -- ? - - D-Mannitol -? ? - - Galactose ?? - - - L-Glutamic acid ?? ? ? - 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 DNA G?C content (mol%) 70.8 65.9 66.2 70.0 69.2 Taxa: 1, YIM 78140T (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 (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 Extraction of genomic DNA and PCR amplification of cultivation in shaken flasks by using ISP 2 broth the 16S rRNA gene were performed from strain YIM medium at 45 °C for about 3 days. Cellular fatty 78140T as described previously (Li et al. 2007). The acid analysis was performed by using the Microbial resulting 16S RNA gene sequence was compared with Identification System (Sherlock Version 6.1; MIDI available 16S rRNA gene sequences of cultured database: TSBA6). The quinones were extracted by from GenBank via the BLAST program and using the method of Collins et al. (1977) and the EzTaxon-e server (http://eztaxon-e.ezbiocloud. separated by HPLC (Kroppenstedt 1982). The G?C net/; Kim et al. 2012). Multiple alignments with DNA content of strain YIM 78140T was deter- sequences of the most closely related taxa and calcu- mined by using the HPLC method of Mesbah et al. lations of levels of sequence similarity were carried (1989). out by using CLUSTAL_X (Thompson et al. 1997).

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Phylogenetic analyses were performed by using three content of the DNA of strain YIM 78140T was tree-making algorithms via the software packages 70.8 mol%, which was slightly higher than those MEGA version 5.0 (Tamura et al. 2011) with three values for the other members of the family Comamo- algorithms, the neighbour-joining (Saitou and Nei nadaceae (65.9–70.0 mol%). The chemotaxonomic 1987), maximum-likelihood (Felsenstein 1981) and characteristics of strain YIM 78140T, such as ubiqui- maximum-parsimony (Fitch 1971) methods. Kimura’s nones and major fatty acids were consistent with its two parameter model was used to calculate Evolu- assignment to the family Comamonadaceae. tionary distance matrices of the neighbour-joining The almost complete 16S rRNA gene sequence of method (Kimura 1980). Bootstrap analysis (1,000 re- strain YIM 78140T (1,548 bp) was determined and samplings) was used to evaluate the topology of phylogenetic analysis indicated that strain YIM phylogenetic trees (Felsenstein 1985). 78140T was related to members of the family Com- amonadaceae in the class b-Proteobacteria and exhibited highest 16S rRNA gene sequence similarity Results and discussion to Caldimonas members of C. manganoxidans JCM 10698T (96.8 %); C. taiwanensis On1T (96.8 %); The cells of strain YIM 78140T were aerobic, Gram- Schlegelella members of S. thermodepolymerans staining negative. Transmission electron micrograph K14T (96.2 %); S. aquatica wcf1T (96.3 %). A total of strain YIM 78140T showed that the cells were short- of 32 nearly full-length 16S rRNA gene sequences rod-shaped and with one single flagellum (Fig. S1). from reference strains were aligned with that of strain 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 Table 2 Comparison of cellular fatty acid compositions (%) 0–3 % (w/v) NaCl. The strain was positive for of strain YIM 78140T and related taxa of the family oxidase, catalase, alkaline phosphatase, esterase, Comamonadaceae esterase lipase, leucine arylamidase, valine arylami- Fatty acids 1 2345 dase, cystine arylamidase, trypsin, acid phosphatase and naphthol-AS-BI-phosphohydrolase, but negative C9:0 3-OH – 1.1 ND – – for urease, amylase, cellulose. Tests of milk coagula- C10:0 – – 1.3 1.2 tion, milk peptonization, hydrolysis of Tweens 20, 40 C10:0 3-OH 4.5 11.1 6.3 3.3 5.1 are positive, but gelatin, pectin and H2S production are C12:0 2.9 7.5 4.3 1.6 2.3 negative. The detailed physiological and biochemical C12:0 3-OH ND – ND 2.0 ND characteristics are listed in the new species descrip- C13:0 – 1.2 – ND ND T tion, and the difference among strain YIM 78140 and C14:0 3.2 5.7 2.9 ND ND its closely related phylogenetic neighbors are shown in C15:1 x6c 2.3 2.3 – ND ND

Table 1. C16:0 19.7 20.5 30.4 43.1 48.7

The analyses indicated that the major cellular fatty C16:1 x5c 2.6 1.5 – ND ND T acid profile of strain YIM 78140 was composed of C17:0 1.9 4.1 – ND ND

C16:0 (19.7 %), C17:0 cyclo (31.0 %), C18:1 x7c C17:0 cyclo 31.0 16.8 1.9 32.6 16.5

(11.3 %) and summed feature 3 (15.5 %). Strain C18:0 – – 2.1 – 1.0 YIM 78140T could be distinguished easily from the C18:1 x7c 11.3 12.0 20.0 4.4 4.3 Schlegelella members of Schlegelella thermodep- Summed feature 3a 15.5 13.0 31.3 – 19.5 olymerans K14T; Schlegelella aquatica wcf1T; Caldi- Taxa: 1, YIM 78140T (data from this study); 2, Caldimonas monas members of Caldimonas manganoxidans JCM T T T manganoxidans JCM 10698 (data from this study); 3, 10698 ; Caldimonas taiwanensis On1 based on the Caldimonas taiwanensis On1T (data from this study); 4, T presence/absence and amount of C14:0,C15:1 x6c, Schlegelella thermodepolymerans K14 ;5,Schlegelella aquatica wcf1T (data from previous study). ‘‘–’’ cellular fatty C16:0 C17:0 cyclo and C18:1 x7c. The comparison of acid values of less than 1 %, ND not detected cellular fatty acid compositions of the novel strain a YIM 78140T and related species of the family Summed features are groups of two or three fatty acids that cannot be separated by GLC with the MIDI system. Summed Comamonadaceae were listed in Table 2. The quinine feature 3 comprises C16:1 x7c and/or C16:1 x6c. Cells were system was composed of Q-8 and Q-9. The G?C harvested after growth on ISP 2 medium at 45 °C for 3 days 123 Antonie van Leeuwenhoek (2014) 105:755–761 759

Leptothrix mobilis Feox-1T (X97071) 63 77 Leptothrix cholodnii CCM 1827 (X97070) 95* 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) 98* Schlegelella aquatica wcf1T (DQ417336) Schlegelella thermodepolymerans K14T (AY152824) T 62* Zhizhongheella caldifontis YIM 78140 (KF771277 ) T 63* Caldimonas manganoxidans JCM 10698 (AB008801) 100* Caldimonas taiwanensis On1T (AY845052) 100 Hydrogenophaga spp. 100* Tepidimonas spp. 90 Burkholderia cepacia Ballard 717T (U96927)

0.01

Fig. 1 Neighbour-joining phylogenetic tree based on 16S the branch points. Burkholderia cepacia Ballard 717T was used rRNA gene sequence (1,548 bp) of strain YIM 78140T and the as an outgroup. Asterisks denote nodes that were also recovered other members of b-Proteobacteria. Bootstrap values (expressed using the maximum-parsimony and maximum-likelihood meth- as percentages of 1,000 replications) of above 50 % are shown at ods. Bar 0.01 substitutions per nucleotide position

YIM 78140T for phylogenetic analysis. It is apparent 78140T, it has the ability to grow well at pH 9.0 and from the neighbour-joining phylogenetic tree (Fig. 1) positive for glucose fermentation, and with relatively that strain YIM 78140T formed a distinct clade with higher DNA G?C content. Therefore, we propose that members of genera Caldimonas and Schlegelella,of strain YIM 78140T should be considered to represent a which was supported by a high bootstrap value of novel species within a new genus, for which the name 100 %. The same affiliation among strain YIM 78140T Zhizhongheella caldifontis gen. nov., sp. nov. is and its closest neighbors C. manganoxidans JCM proposed. 10698T, C. taiwanensis On1T, S. thermodepolymerans K14T and S. aquatica wcf1T was also supported by Description of Zhizhongheella gen. nov. maximum parsimony and maximum-likelihood algo- rithms with bootstrap values of 100.0 and 99.0 %, Zhizhongheella [Zhi.zhong.he.el’la. N.L. fem. dim. respectively (Supplementary Figs. S2, S3). Therefore, N. Zhizhongheella named after Zhi-Zhong He strain YIM 78140T may be considered as a different (1939–2012), a Chinese microbiologist who devoted genomic genus in the family Comamonadaceae. himself to the biology of thermophiles]. Besides the phylogenetic analysis based on 16S Cells are aerobic, thermotolerant, short-rod-shaped, rRNA gene sequences, strain YIM 78140T could also and Gram-stain negative. Growth occurs at 10–55 °C be differentiated readily from C. manganoxidans JCM and pH 6.0–10.0, with optimum growth at 40–50 °C 10698T,C.taiwanensis On1T,S.thermodepolymerans and pH 8.0–9.0, respectively. The predominant ubiq- K14T and S. aquatica wcf1T based on many pheno- uinones are Q-8 and Q-9. Major fatty acids ([10 %) typic and chemotaxonomic characteristics shown in are C16:0,C17:0 cyclo, C18:1 x7c and summed feature 3 Tables 1 and 2. Particularly for the new isolate (C16:1 x7c and/or C16:1 x6c). The G?C content of

123 760 Antonie van Leeuwenhoek (2014) 105:755–761 genomic DNA of the type strain of the type species is Acknowledgments The authors are grateful to Prof. Dr. Takuji 70.8 mol%. The type species is Zhizhongheella Kudo (JCM) and Mrs. Min Tseng (BCRC) for their kind providing reference type strains. This research was supported by caldifontis. the Key Project of International Cooperation of Ministry of Science & Technology (MOST) (No. 2013DFA31980), National Science Foundation grant (OISE-0968421) and Key Project of Description of Zhizhongheella caldifontis sp. nov. Yunnan Provincial Natural Science Foundation (2013FA004).

Zhizhongheella caldifontis (cal.di’fon’tis. L. adj. cal- dus, hot; L. n. fons fontis, a spring; N.L. gen. N. caldifontis, of a hot spring). References In addition to the characteristics given in the genus Blackall LL, Crocetti GR, Saunders AM, Bond PL (2002) A description above, exhibits the following characteris- review and update of the microbiology of enhanced bio- tics. Good growth is observed on ISP 2 and modified logical phosphorus removal in wastewater treatment T5 media, colonies on ISP 2 medium are circular, plants. Antonie Van Leeuwenhoek 81:681–691 convex and light brown. Growth occurs in the Cerny G (1978) Studies on aminopeptidase for the distinction of Gram-negative from Gram-positive . Appl Micro- presence of 0–3 % (w/v) NaCl. Positive for oxidase, biol Biotechnol 5:113–122 catalase, alkaline phosphatase, esterase, esterase Chou YJ, Sheu SY, Sheu DS, Wang JT, Chen WM (2006) lipase, leucine arylamidase, valine arylamidase, cys- Schlegelella aquatica sp. nov., a novel thermophilic bac- tine arylamidase, trypsin, acid phosphatase and terium isolated from a hot spring. Int J Syst Evol Microbiol 56:2793–2797 naphthol-AS-BI-phosphohydrolase, but negative for Collins MD, Pirouz T, Goodfellow M, Minnikin DE (1977) urease, amylase, cellulose. Tests for milk coagulation, Distribution of menaquinones in actinomycetes and cory- milk peptonization, hydrolysis of Tweens 20, 40 are nebacteria. J Gen Microbiol 100:221–230 positive, but gelatin, pectin and H S production are Dubinina GA, Grabovich MY (1984) Isolation, cultivation, and 2 characteristics of bipunctata. Microbiology negative. Acetate, adipate, bromo-succinic acid, citric (English translation of Microbiologia) 53:610–617 acid, dextrin, D-cellobiose, D-turanose, glycerol, D- Elbanna K, Lu¨tke-Eversloh T, van Trappen S, Mergaert J, glucose-6-PO4, D-fructose-6-PO4, D-xylose, D-arabi- Swings J, Steinbu¨chel A (2003) Schlegelella thermodep- olymerans gen. nov., sp. nov., a novel thermophilic bac- nose, D-glucose, D-gluconic acid, D-saccharic acid, terium that degrades poly(3-hydroxybutyrate-co-3- D-lactic acid methyl ester, lactose, L-lactic acid, mercaptopropionate). Int J Syst Evol Microbiol 53: galactose, gluconate, maltose, malate, methyl pyru- 1165–1168 vate, mycose, p-hydroxy-phtnylacetic acid, raffinose, Felsenstein J (1981) Evolutionary trees from DNA sequences: a sorbose and a-keto-glutaric acid can be utilized as maximum likelihood approach. J Mol Evol 17:368–376 Felsenstein J (1985) Confidence limits on phylogenies: an sole carbon sources, but adonitol, capric acid, dulci- approach using the bootstrap. Evolution 39:783–791 tol, D-trehalose, D-mannose, D-galactose, D-fructose, Fitch WM (1971) Toward defining the course of evolution: D-fucose, D-glucuronic acid, inositol, L-rhamnose, minimum change for a specific tree topology. Syst Zool 20:406–416 L-arabinose, L-xylose, sorbitol, mannitol, mucic acid, Ginige MP, Hugenholtz P, Daims H, Wagner M, Keller J, phenylacetate, sucrose and xylitol are not utilized. Blackall LL (2004) Use of stable-isotope probing, full- Glucuronamide, L-glutamic acid, L-pyroglutamic acid, cycle rRNA analysis, and fluorescence in situ hybridiza- N-acetyl-b-D-mannosamine are utilized as solo nitro- tion-microautoradiography to study a methanol-fed deni- trifying microbial community. Appl Environ Microbiol gen sources, but not L-alanine, L-arginine, L-aspartic 70:588–596 acid, L-histidine and L-serine. Acid is produced Gonzalez C, Gutierrez C, Ramirez C (1978) Halobacterium from D-arabinose, fructose, glucose, lactose, mycose, vallismortis sp. nov., an amylolytic and carbohydrate- sorbose and raffinose. metabolizing, extremely halophilic bacterium. Can J The type strain, YIM 78140T (= BCRC Microbiol 24:710–715 T T Grabovich M, Gavrish E, Kuever J, Lysenko AM, Podkopaeva 80649 = KCTC 32557 ) was isolated from a hot D, Dubinina G (2006) Proposal of Giesbergeria voro- spring water sample (N 24.9728°, W 98.39706°) from nezhensis gen. nov., sp. nov. and G. kuznetsovii sp. nov. and Hehua hot spring, Tengchong, Yunnan province, reclassification of [Aquaspirillum] anulus,[A.] sinuosum south-west China. The 16S rRNA gene sequence of and [A.] giesbergeri as Giesbergeria anulus comb. nov., G. T sinuosa comb. nov. and G. giesbergeri comb. nov., and strain YIM 78140 has been deposited in GenBank [Aquaspirillum] metamorphum and [A.] psychrophilum as under the accession number KF771277. Simplicispira metamorpha gen. nov., comb. nov. and S.

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