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 philippinense 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-diaminopimelic 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. (2007). The 16S rRNA gene sequence of strain KLBMP 1111T was compared against a database via BLAST Results and discussion analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and the EzTaxon server (http://www.eztaxon.org) (Chun Strain KLBMP 1111T is an aerobic actinomycete that et al. 2007) of type strains to retrieve most similar forms extensively branched substrate mycelia and sequences of recognized species. Multiple alignments grew well on most of the test media. Moderate growth were performed using the CLUSTAL_X program occurred on ISP 3 and ISP 4 media, while weak growth (Thompson et al. 1997). The phylogenetic tree was was observed on Czapek’s agar. Black diffusible constructed with the neighbour-joining (Saitou and Nei pigments were produced on ISP 2 and nutrient agar. 1987) and maximum-parsimony (Kluge and Farris White to brown substrate mycelia and white or gray– 1969) methods using the software package MEGA white aerial mycelia, with spores arranged in long version 4.1 (Tamura et al. 2007) and distances were straight or curved chains were produced on all media calculated according to Kimura’s two-parameter tested. Many sporangium-like globular bodies were model (Kimura 1980). Bootstrap analysis was used to also found on aerial mycelia but release of spores from evaluate the tree topology of the neighbour-joining data these was not observed (Fig. 1). Good growth by performing 1000 resamplings (Felsenstein 1985). occurred at 28–30°C and at pH 7–7.5 with 0–1% (w/ DNA–DNA hybridization was determined using v) NaCl. Detailed results of the physiological and the photobiotin-labelled probes method in microplate biochemical analyses are given in Table 1 and in the 123 436 Antonie van Leeuwenhoek (2012) 101:433–441 species description. It is evident from Table 1 that phosphatidylglycerol, phosphatidylinositol and an there are several phenotypic characteristics that unknown phospholipid (type PP phospholipid pattern, clearly separate strain KLBMP 1111T from recog- Lechevalier et al. 1977, Fig. S1). The major fatty acids nized Kibdelosporangium species. present were iso-C16:0 (28.8%), C16:0 (12.9%), anteiso- T The assignment of strain KLBMP 1111 to the C15:0 (10.8%), iso-C15:0 (9.4%), anteiso-C17:0 (6.8%) genus Kibdelosporangium is also supported by the and iso-C14:0 (6.6%). This fatty acid profile is similar to chemotaxonomic data. The cell-wall peptidoglycan the three recognized Kibdelosporangium species and contained meso-diaminopimelic acid as a diagnostic there are differences in the proportions of some fatty amino acid and the whole-cell sugars comprised acids. A detailed fatty acid profile comparison is given arabinose and galactose (type IV cell walls) (Lecheva- in Table 2. The G?C content of the DNA of the type lier and Lechevalier 1970). Mycolic acids were not strain is 67.2 mol%. present. The predominant menaquinone was MK-9(H4) The 16S rRNA gene sequence comparison clearly T (76.7%) with MK-9(H2) (9.5%), MK-9(H6) (6.5%) and showed that strain KLBMP 1111 belongs to the MK-9(H8) (7.3%) as minor components. The phospho- genus Kibdelosporangium in the actinobacterial fam- lipid profile consisted of phosphatidylethanolamine, ily Pseudonocardineae and formed a distinct subclade phosphatidylmethylethanolamine, in the phylogenetic cluster of the genus Kibdelospo- rangium with high bootstrap support (Fig. 2). 16S rRNA gene sequence similarities between strain KLBMP 1111T and the type strains of K. aridum subsp. aridum DSM 43828T, K. aridum subsp. largum DSM 44150T and K. philippinense DSM 44226T were 98.8, 98.1 and 98.1%, respectively. However, DNA– DNA hybridization experiments revealed that the relatedness between strain KLBMP 1111T and K. aridum subsp. aridum DSM 43828T (38.4 ± 1.2%), K. aridum subsp. largum DSM 44150T (49.9 ± 3.5%) and K. philippinense DSM 44226T (32 ± 3.7%) were all well below the 70% cut-off value generally recognized for genomic species (Stackebrandt and Goebel 1994), suggesting that strain KLBMP 1111T should be considered as a novel species. Strain KLBMP 1111T could not growth on nitrogen-free NFb semisolid medium and did not produce IAA. However, siderophore production was detected by the formation of orange halos surrounding colony on CAS agar plates after 48 h incubation at 28°C. Strain KLBMP 1111T also showed growth in ADF medium suggesting that it possessed ACC deaminase enzyme activity. By quantitative determination, strain KLBMP 1111T exhibited high ACC deaminase activity (0.092 lmol a-ketobutyrate mg-1 h-1). It has been reported that greater or equal to 20 nmol a-ketobutyrate mg-1 h-1 ACC is sufficient to show plant growth promoting effects (Penrose and Glick 2003). Thus, because of the ability to produce siderophores and ACC deaminase, strain KLBMP 1111T may have the potential to promote plant growth. Fig. 1 Scanning electron micrographs of strain KLBMP 1111T grown on ISP 2 medium for 28 days at 28°C. a Aerial mycelia The combination of phylogenetic, phenotypic and and spore chains; b a sporangiumlike globular body chemotaxonomic data demonstrate that strain KLBMP 123 Antonie van Leeuwenhoek (2012) 101:433–441 437

Table 1 Differential characteristics of strain KLBMP 1111T and the species of the genus Kibdelosporangium Characteristic KLBMP K. aridum subsp. K. aridum subsp. K. philippinense 1111T aridum DSM 43828T largum DSM 44150T DSM 44226T

Growth on ISP 4 medium: Moderate Poor Poor Poor Growth on ISP 5 medium: Good Poor Moderate Good Formatin of aerial mycelia on ISP 2 Abundant Sparse Sparse Abundant and ISP 3 media: Diffusible pigment Black Light brown None None Growth at 158C ?- ? ? Growth at 458C - w ?- Growth on 6% NaCl -? ? ? Utilization of: D-Arabinose ? w ?- D-Rafﬁnose ?? ? - a-Lactose ?? - ? D-Ribose -? ? - Inositol -? - - D-Sorbitol ?? - - Sucrose -? ? - Xylitol -? - - D-Rhamnose -- ? - Trehalose ?? - ? D-Fructose ?- ? ? D-Valine ?? - - L-Histidine ?- ? - L-Arginine ?? - - Reduction of nitrate -- - ?

H2S production ?- - - Note ? Positive or present; w weakly positive; - negative or absent. All the data were obtained from this study under identical growth conditions

1111T is a member of the genus Kibdelosporangium.It Description of K. phytohabitans sp. nov could, however, be readily differentiated from the type strains of K. aridum subsp. aridum DSM 43828T, Kibdelosporangium phytohabitans (Phy.to.habitans. K. aridum subsp. largum DSM 44150T and K. philip- Gr. n. phyton, plant; L. part. adj. habitans, inhabiting; pinense DSM 44226T based on several phenotypic N. L. part. adj. used as a masc. n. phytohabitans, plant characteristics (Table 1); e.g. different growth charac- inhabiting, isolated from a plant). teristics on ISP 4 and ISP 5 media, formation of aerial Gram positive, aerobic, non-motile actinomycete. mycelia on ISP 2/3 media of strain KLBMP 1111T, The aerial mycelium is white or gray–white and the different range for growth with respect to temperature, vegetative mycelium is white to brown. This species pH and NaCl tolerance and the ability to produce H2S. forms extensively branched substrate mycelia. Black Based on the polyphasic taxonomic characterisation diffusible pigment is produced. The aerial mycelium presented above, strain KLBMP 1111T can be proposed formed long, straight or curved chains of cylindrical to represent a new species in the genus Kibdelosporan- spores as well as numerous sporangium-like rugose- gium, for which the name Kibdelosporangium phyto- surfaced globular bodies. Good growth occurs on ISP habitans sp. nov. is proposed. 2, ISP 5, nutrient agar and potato agar media. The pH

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Table 2 Fatty acid proﬁles (%) of strain KLBMP 1111T and related Kibdelosporangium species Fatty acid 1 2 3 4 iso-C12:0 – 0.1 0.1 0.1

C12:0 – 0.1 – 0.9

C12:0 3 OH – – – 0.4 iso-C13:0 0.1 0.1 0.1 0.1 anteiso-C13:0 – – 0.1 0.1

C13:0 0.1 0.2 0.1 0.1

C14:0 3.1 2.1 3.4 3.4 iso-C14:0 6.6 10.1 2.7 1.8 anteiso-C14:0 0.1 – 0.1 –

C14:1 x5c – 0.1 0.1 0.1 iso-C15:0 9.4 4.9 13.0 13.3 anteiso-C15:0 10.8 3.5 14.7 6.9

C15:1 x6c – – 0.7 –

C15:0 2 OH – 0.5 – 0.1 iso-C16:0 28.8 33.9 24.7 20.7 anteiso-C16:0 0.3 0.3 0.7 1.2

C16:0 12.9 4.1 4.7 5.4 iso-C16:1 H 1.7 6.5 2.7 4.1

C16:0 3 OH – 0.1 – – iso-C17:0 1.5 0.5 1.5 1.8 anteiso-C17:0 6.8 1.7 12.3 7.9

C17:0 3.1 2.0 1.3 1.9

C17:0 3 OH 0.2 0.5 0.1 –

C17:0 2 OH – – 0.1 –

C17:0 10-methyl – 0.2 0.4 anteiso-C17:1x9c – 0.2 0.3

C17:1x8c 0.7 1.1 0.2 1.2

C17:1x6c 4.8 14.1 8.0 9.5 iso-C18:0 0.3 0.3 0.3 0.3 iso-C18:1 H––––

C18:1x9c 0.1 3.9 – 2.0

C18: 0 3 OH 0.3 0.3 0.2 0.3

C19:0 cyclo x8c – – 0.1 0.1 Sum in feature 3a 6.8 3.1 6.5 12.1 Sum in feature 5a – 4.7 0.1 – Sum in feature 9a 1.6 1.5 1.6 3.4 Strains 1, KLBMP 1111T;2,K. philippinense DSM 44226T;3,K. aridum subsp. aridum DSM 43828T;4,K. aridum subsp. largum DSM 44150T. All the data are from this study. Values are percentages of total fatty acids; – not detected a Summed features represent groups of two or three fatty acids that cannot be separated by GC with the MIDI system. Summed features 3, 5, 9 comprised 16:1 w7c/16:1 w6c, 18:2 w6,9c/18:0 ante, 17:1 iso w9c/16:0 10-methyl, respectively range for growth is 6.0–8.0, with an optimum pH D-fructose, D-galactose, D-glucose, glycerol, lactose, 7.0. The temperature range for growth is 15–37°C, maltose, mannitol, mannose, D-rafﬁnose, D-ribose, with optimal growth temperature of 28°C. The sorbitol, sorbose, starch, trehalose and D-xylose are NaCl tolerance is up to 3%. D-Arabinose, cellobiose, used as sole carbon and energy sources, whereas

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Fig. 2 Neighbour-joining tree based on almost complete 16S rRNA gene sequences, showing the relationship between strain KLBMP 1111T and its phylogenetic neighbours. Only bootstrap values above 50%, expressed as percentages of 1000 replications, are shown at the branch points. Asterisks indicate the clades that were conserved when neighbour- joining and maximum- parsimony methods were used to construct phylogenetic trees. Bar 0.005 substitutions per nucleotide position

dextrin, dulcitol, erythritol, inositol, D-ribose, L-rham- Priority Academic Program Development of Jiangsu Higher nose, sucrose and xylitol are not. The cells contain Education Institutions (PAPD), the Project of Outstanding Scientific and Technological Innovation Team for Higher meso-diaminopimelic acid, arabinose and galactose Education Institutions in Jiangsu Province (Pre-development (type IV cell walls), and no mycolic acids. The major of medical microbiology) and Grants from Natural Science menaquinone is MK-9(H4). The phospholipids present Foundation by Xuzhou City (No. XZZD1004) and Xuzhou are phosphatidylethanolamine, phosphatidylmethy- Normal University (09XLR12, 09XLR19). lethanolamine, phosphatidylglycerol, phosphatidylinositol and an unknown phospholipid (type PP phospholipid pattern). Major fatty acids are iso- References C16:0,C16:0, anteiso-C15:0 and iso-C15:0. The G?C content of the DNA is 67.2 mol%. T T Chun J, Lee JH, Jung Y, Kim M, Kim S, Kim BK, Lim YW The type strain, KLBMP 1111 (=KCTC 19775 = (2007) EzTaxon: a web-based tool for the identification of CCTCC AA 2010001T) was isolated from surface- prokaryotes based on 16S ribosomal RNA gene sequences. sterilized roots of Jatropha curcas L. collected from Int J Syst Evol Microbiol 57:2259–2261 Collins MD, Jones D (1980) Lipids in the classification and the city of Panzhihua, Sichuan Province, south-west identification of coryneform bacteria containing peptido- China. glycan based on 2,4-diaminobutyric acid. J Appl Bacteriol 48:459–470 Acknowledgments This research was partially supported by Collins MD, Pirouz T, Goodfellow M, Minnikin DE (1977) National Natural Science Foundation of China (Nos. 31000005, Distribution of menaquinones in actinomycetes and cory- 31101502), the Program of Natural Science Foundation of the nebacteria. J Gen Microbiol 100:221–230 Jiangsu Higher Education Institutions of China (Nos. Doebereiner J (1995) Isolation and identification of aerobic 11KJD210002, 10KJB180008), the Project Funded by the nitrogen-fixing bacteria from soil and plants. In: Alef K, 123 440 Antonie van Leeuwenhoek (2012) 101:433–441

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