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Kibdelosporangium Phytohabitans Sp. Nov., a Novel Endophytic Actinomycete Isolated from Oil-Seed Plant Jatropha Curcas L

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Kibdelosporangium Phytohabitans Sp. Nov., a Novel Endophytic Actinomycete Isolated from Oil-Seed Plant Jatropha Curcas L Antonie van Leeuwenhoek (2012) 101:433–441 DOI 10.1007/s10482-011-9652-4 ORIGINAL PAPER Kibdelosporangium phytohabitans sp. nov., a novel endophytic actinomycete isolated from oil-seed plant Jatropha curcas L. containing 1-aminocyclopropane- 1-carboxylic acid deaminase Ke Xing • Guang-Kai Bian • Sheng Qin • Hans-Peter Klenk • Bo Yuan • Yue-Ji Zhang • Wen-Jun Li • Ji-Hong Jiang Received: 23 July 2011 / Accepted: 30 September 2011 / Published online: 12 October 2011 Ó Springer Science+Business Media B.V. 2011 Abstract A novel actinomycete, designated strain formed a distinct branch in the 16S rRNA gene KLBMP 1111T, was isolated from the root of the oil- phylogenetic tree together with the type strains in the seed plant Jatropha curcas L. collected from Sichuan genus Kibdelosporangium, with the highest similarity Province, south-west China. Strain KLBMP 1111T to Kibdelosporangium aridum subsp. aridum DSM 43828T (98.8%), K. aridum subsp. largum DSM 44150T (98.1%) and Kibdelosporangium philippin- ense DSM 44226T (98.1%). The organism produced sporangium-like structures, the typical morphological characteristic of the genus Kibdelosporangium. The chemotaxonomic properties of this strain were also Electronic supplementary material The online version of consistent with those of the genus Kibdelosporan- this article (doi:10.1007/s10482-011-9652-4) contains supplementary material, which is available to authorized users. gium: the peptidoglycan contained meso-diamino- pimelic acid; the predominant menaquinone was K. Xing Á G.-K. Bian Á S. Qin (&) Á B. Yuan Á MK-9(H ); phospholipids were phosphatidylglycerol, & 4 Y.-J. Zhang Á J.-H. Jiang ( ) phosphatidylethanolamine, phosphatidylmethyletha- School of Life Science, The Key Laboratory of Biotechnology for Medicinal Plant of Jiangsu Province, nolamine, phosphatidylinositol and an unknown Xuzhou Normal University, Xuzhou, phospholipid; iso-C16:0,C16:0, anteiso-C15:0 and iso- Jiangsu 221116, People’s Republic of China C15:0 as the predominant cellular fatty acids and the e-mail: [email protected] G?C content was 67.2 mol%. DNA–DNA hybrid- J.-H. Jiang ization values between strain KLBMP 1111T and the e-mail: [email protected] three Kibdelosporangium species were less than 50%. H.-P. Klenk This strain had the ability to produce a siderophore, DSMZ, Deutsche Sammlung von Mikroorganismen und utilized 1-aminocyclopropane-1-carboxylic acid Zellkulturen GmbH, Inhoffenstraße 7b, 38124 (ACC) as sole source of nitrogen and possessed Braunschweig, Germany ACC deaminase enzyme. Based on genotypic and T W.-J. Li phenotypic data, strain KLBMP 1111 represents a The Key Laboratory of Microbial Diversity novel species in the genus Kibdelosporangium.We in Southwest China, Ministry of Education propose the name Kibdelosporangium phytohabitans and Laboratory for Conservation and Utilization sp. nov. for this species. The type strain is the strain of Bio-Resources, Yunnan Institute of Microbiology, T T Yunnan University, Kunming 650091, KLBMP 1111 (=KCTC 19775 = CCTCC AA People’s Republic of China 2010001T). 123 434 Antonie van Leeuwenhoek (2012) 101:433–441 Keywords Kibdelosporangium phytohabitans sp. Materials and methods nov. Á Polyphasic taxonomy Á 16S rRNA Á 1-aminocyclopropane-1-carboxylic acid Isolation of organism Healthy root samples of Jatropha curcas L., an oil- seed traditional Chinese medicinal plant, collected Introduction from the city of Panzhihua, Sichuan Province, south- west China were used as the source for isolation of Actinomycetes are widely distributed in terrestrial and endophytic actinomycetes. Samples were air dried for aquatic ecosystems and studies of rare actinomycetes 48 h and subsequently washed ultrasonically to thor- may result in increased chances of discovering novel oughly remove the surface soils. The roots were then lead compounds (Tiwari and Gupta 2011). Endophytic surface sterilized according to the procedures of Qin actinomycetes, which exist in the inner tissues of et al. (2008). Subsequently, the surface sterilized living plants, have attracted increasing attention in samples were aseptically crumbled into smaller frag- recent years. Numerous studies have indicated that ments using a commercial blender (Joyoung), spread these organisms have the capacity to produce an onto glycerol-asparagine (ISP 5) agar medium (Shir- impressive array of secondary metabolites exhibiting a ling and Gottlieb 1966) and incubated at 28°C for wide variety of biological activity, such as antibiotics, 2–6 weeks. Strain KLBMP 1111T was picked after antitumour and anti-infection agents and plant growth incubation for 28 days. The purified isolate was promoters that may contribute to their host plants by routinely cultured on yeast extract-malt extract agar promoting growth and enhancing their ability to (ISP 2) (Shirling and Gottlieb 1966) and maintained as withstand environmental stresses (Strobel and Daisy a glycerol suspension (20%, w/v) at -80°C. 2003; Qin et al. 2011). These organisms have large biodiversity and to date, more than 50 new taxa have Phenotypic characterization been found and the majority of which are rare actinobacteria (Qin et al. 2011). Cultural characteristics of the new isolate were The members of the genus Kibdelosporangium are determined after 2 weeks at 28°C on the media known to produce novel antibiotics with glycopeptide, described by Shirling and Gottlieb (1966) as well as macrolides and polyketide structures (Tiwari and potato-dextrose agar, Czapek’s agar and nutrient agar Gupta 2011). The genus was originally proposed by (Waksman 1967). National Bureau of Standards Shearer et al. (1986a) to accommodate actinomycetes (NBS) Colour Name Charts (Kelly 1964) were used that produce both long chains of aerial spores and for determining colour designation and names. Mor- sporangium-like structures. Members of the genus phological characteristics were observed using light contain meso-diaminopimelic acid as the diagnos- microscopy (SA3300-PL) and scanning electron tic diamino acid, type PP phospholipids (the predom- microscopy (Hitachi; S-3400 N) after 4 weeks growth inant polar lipid is phosphatidylethanolamine), on ISP 2 medium agar. Growth at different temper- MK-9(H4) as the major menaquinone and no mycolic atures (4, 10, 15, 20, 25, 28, 30, 37, 40, 45, 50°C), acids. At the time of writing, the genus Kibdelospo- different NaCl concentrations (0–15% w/v at intervals rangium comprised only three taxa: Kibdelosporan- of 0.5%) and at various pH values (pH 5.0–12.0 at gium aridum subsp. aridum DSM 43828T, K. aridum intervals of 0.5 pH unit with different pH buffers) at subsp. largum DSM 44150T (Shearer et al. 1986b) and 28°C was tested on ISP 2 basal medium after 14 days K. philippinense DSM 44226T (Mertz and Yao 1988). of incubation as described by Xu et al. (2005). In the present study, strain KLBMP 1111T was Utilization of sole carbon sources for energy and isolated from the oil-seed medicinal plant Jatropha growth, and decomposition of test substances was curcas L. during an investigation of actinomycete carried out according to Kurup and Schmitt (1973) and diversity from medicinal plants. The aim of the present Gordon et al. (1974). Three type strains, K. aridum study was to establish the taxonomic position of this subsp. aridum DSM 43828T, K. aridum subsp. largum organism and the results indicate that the strain repre- DSM 44150T and K. philippinense DSM 44226T were sents a novel species in the genus Kibdelosporangium. tested concurrently under the same conditions. 123 Antonie van Leeuwenhoek (2012) 101:433–441 435 Chemotaxonomy wells as described by Ezaki et al. (1989) and He et al. (2005). The hybridizations were carried out with five Biomass for quantitative fatty acid analysis was replications and the hybridization temperature was obtained from cultures grown in tryptic soy broth 45°C after the formamide was added. (TSB) at 150 rpm for 7 days at 28°C. Biomass for other chemotaxonomy studies was obtained by culti- Plant growth-promoting potential evaluation vation in shaken flasks (150 rpm) using ISP 2 medium at 28°C for 7 days. Analysis of cell-wall amino acids Several plant growth-promoting traits for strain and sugars in whole-cell hydrolysates were carried out KLBMP 1111T were tested. Biological nitrogen fixa- according to the methods described by Hasegawa et al. tion ability of the isolate was tested using the acetylene (1983) and Lechevalier and Lechevalier (1970). reduction method in nitrogen-free semisolid NFb Phospholipids were extracted, examined by two- medium (Doebereiner 1995). Siderophore production dimensional TLC and identified by using published was detected by the formation of orange-yellowish procedures (Minnikin et al. 1979; Collins and Jones halos surrounding actinobacterial colonies on chrome 1980). Analysis of mycolic acids was performed using azurol sulfonate (CAS) agar plates after 48 h incuba- the previously described method by Minnikin et al. tion at 28°C (Schwyn and Neilands 1987). Indole acetic (1980). The fatty acids were extracted, purified, acid (IAA) production was determined using the methylated and quantified by GC using the standard colourimetric method described by Gordon and Weber Microbial Identification System (Sherlock version 6.1 (1951). To study the utilization of 1-aminocyclopro- MIDI; TSBA6 database) (Sasser 1990;Ka¨mpfer and pane-1-carboxylic acid (ACC) as sole nitrogen source, Kroppenstedt 1996). Cellular menaquinones were the strain was grown in ADF medium supplemented extracted and purified as described by Collins et al. with 3 mM ACC at 28°C for 5 days (Jacobson et al. (1977) and were analysed by HPLC (Groth et al. 1994). ACC deaminase enzyme activity assay was 1997). Determination of DNA G?C content was carried out according to El-Tarabily (2008). performed according to Mesbah et al. (1989). Nucleotide sequence accession number Molecular analysis The 16S rRNA gene sequence of strain KLBMP 1111T Genomic DNA extraction, PCR amplification of the determined in this study has been deposited in 16S rRNA gene and sequencing of the PCR products GenBank under the accession number HM153787. were carried out as described by Li et al.
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