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Zhizhongheella Caldifontis Gen. Nov., Sp. Nov., a Novel Member of the Family Comamonadaceae

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Zhizhongheella Caldifontis Gen. Nov., Sp. Nov., a Novel Member of the Family Comamonadaceae 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 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-Proteobacteria (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 taxonomy 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). Enzyme 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. 123 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 ?- - - - 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 species 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.
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