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International Journal of Systematic and Evolutionary Microbiology (2013), 63, 893–899 Pseudonocardia antitumoralis sp. nov., adeoxynyboquinone-producing actinomyceteisolated from a deep-sea sediment Xin-Peng Tian,1 Li-Juan Long,1 Su-Mei Li,1 Jing Zhang,1 Ying Xu,2Jie He,2 Jie Li,1 Fa-Zuo Wang,1 Wen-Jun Li,2 Chang-Sheng Zhang1and Si Zhang1 1Key Laboratory of Marine Bio-resources Sustainable Utilization, CAS; RNAM Center for Marine Microbiology, CAS; Guangdong Key Laboratory of Marine Materia Medica; South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, 510301, PR China 2Key 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, Yunnan, 650091, PR China An aerobic actinomycete, designated SCSIO 01299T, was isolated from a deep-sea sedimentcollected from the northern South China Sea at a depth of 3258 m. The isolate was found to be anatural producer of the synthesized antitumour agent deoxynyboquinone and its three newderivatives, pseudonocardians A, B and C. A BLAST search based on almost-complete 16S rRNAgene sequences showed that strain SCSIO 01299T had high sequence similarities with membersof the genus Pseudonocardia. The 16S rRNA gene sequence phylogenetic tree revealed thatstrain SCSIO 01299T was a member of the genus Pseudonocardia. Phenotypic analysis,chemotaxonomy and DNA–DNA relatedness could readily distinguish the isolate from establishedmembers in this genus. It was concluded that strain SCSIO 01299T represents a novel species,for which the name Pseudonocardia antitumoralis sp. nov. is proposed. The type strain is SCSIO01299T (5DSM 45322T 5CCTCC M 2011255T).
Actinomycetes are well known as the largest group (45 %) of actinomycetal taxa is very important for gaining new sources bioactive secondary metabolite producers of pharmaceutical potential.
Over 50 years of research on terrestrial actinomycetes, the rate of discovery of new compounds has decreased because resources in the South China Sea, a deep-sea sediment of the high rate of reisolation of producers of known sample (120u 0.9759 E 19u 0.6649 N) at a depth of 3258 m compounds ). For the discovery of (pH 7.8, 2 uC) was collected. The sampling process and structurally diverse natural products in drug discovery isolation procedure were done as described by programmes, novel screening procedures and high-quality One grey-white aerial mycelial colony with an biological materials from new sources are two critical factors obvious inhibition zone after 3 weeks at 28 uC on selective (). So, extremophiles have been isolation medium ISP 5 prepared with natural seawater, focused on increasingly by chemists, including actinomyce- designated strain SCSIO 01299T, was selected. By the tal resources in marine environments. Recent studies have bioactivity-guided isolation methods, we found that the revealed that marine actinomycetes are still a robust source isolate could produce deoxynyboquinone (15 mg l21), a of excellent natural products, such as the genera Salinispora synthetic potential antitumour agent, and its three new salinosporamide A (NPI-0052), sporolides, saliniquinone derivatives, pseudonocardians A (0.66 mg l21), B (1.5 mg A-F, salinosporamide K), Verrucosispora (abyssomicins), l21) and C (1.3 mg l21) Strain SCSIO Micromonospora [diazepinomicin (ECO-4601)] 01299T was maintained on ISP 2 or stored at 280 uC as a and ‘Marinispora' (marinomycins, marinisporolides) ( 20 % (w/v) glycerol suspension.
Therefore, the discovery of novel marine Morphological observation and cultural characteristics The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene were performed on Difco media ISP 2, 3, 4 and 5, sequence of strain SCSIO 01299T is JN204514.
Czapek's agar nutrient agar and potato Two supplementary figures are available with the online version of this agar at 28 uC for 28 days. Cellular morphology was examined by light microscopy and scanning electron 037135 G 2013 IUMS Printed in Great Britain

X.-P. Tian and others Table 1. Culture characteristics of strain SCSIO 01299T and its as described by Catalase activity was closest phylogenetic neighbours in the genus Pseudonocardia evaluated by the production of oxygen bubbles in 3 % (v/v)H2O2 and oxidase activity was determined using oxidase Strains: 1, Pseudonocardia antitumoralis sp. nov. SCSIO 01299T; 2, P.
reagent (bioMe´rieux). Growth at 4, 10, 15, 20, 28, 35, 40, ammonioxydans CGMCC 4.1877T; 3, P. kongjuensis DSM 44525T; 4, P.
45 and 50 uC, at pH 4.5–11 (in increments of 0.5 pH unit) autotrophica BCRC 12444T; 5, P. endophytica YIM 56035T. All data and with 0–25 % (w/v) NaCl (at intervals of 2.5 %) was were taken from this study after incubation at 28 uC for 21 days.
studied as described by The buffer Colours were taken from ISCC–NBS colour charts All solutions used to adjust the pH were acetate/citrate (pH 4– strains form white aerial mycelium on ISP 2, 3 and 4. None of the 6), KH2PO4/NaOH (pH 6–8), NaHCO3/Na2CO3 (pH 9– strains forms soluble pigment on ISP 2, 3, 4 and 5, nutrient agar or 10) and Na2HPO4/NaOH (pH 11). Sole carbon source potato agar. DOY, Dark orange yellow; LOY, light orange yellow; MOY, utilization tests (final concentration 0.5 %, w/v) were moderate orange yellow; MYB, moderate yellowish brown; POY, pale performed as described by orange yellow; PW, pinkish white; W, white; YW, yellowish white; 2, no Antibiotic susceptibility was examined as described by using antibiotic discs on ISP 2.
Biomass for chemotaxonomic and molecular systematicstudies was obtained at the same physiological age by Substrate mycelium cultivation in ISP 2 with shaking at 150 r.p.m. at 28 uC for Substrate mycelium 1 week. Cell-wall diaminopimelic acid and whole-cell Substrate mycelium sugars, menaquinones and phospholipids were analysed Substrate mycelium according to previously established procedures Czapek's agar Aerial mycelium ). Cellular fatty acid analysis was Substrate mycelium performed according to the protocol of the Sherlock Microbial Identification System (MIDI) by GC (model Nutrient agar Aerial mycelium 6890; Agilent), based on biomass obtained by culture in Substrate mycelium TSB (Difco) for 4 days at 28 uC. The results were compared with the TSBA 6 database of fatty acids in Sherlock version Substrate mycelium 6.1. The G+C content of genomic DNA was determinedusing the HPLC method Extraction of genomic DNA and PCR amplification of 16S microscopy (JSM 5600LV; JEOL) using cells incubated for rRNA gene were carried out according to 14 and 28 days on ISP 2. Diffusible pigments were Similarity calculations were performed based on EzTaxon-e observed by comparing the cultures with the most suitable and MEGA version 5.0 colour chips from the ISCC-NBS colour charts with parameters of pairwise deletion for gap treat- All physiological and biochemical tests were ment. Multiple alignments were created using CLUSTAL X performed at 28 uC, such as diffusible pigment, hydrolase Phylogenetic trees were con- and H2S and melanin production, according to methods structed using MEGA version 5.0 with the neighbour-joining described by Gram-reaction was done method of with the parameter of Fig. 1. Scanning electron micrograph of aerialmycelia and spore chains of strain SCSIO01299T after incubation for 14 days on ISP 2at 28 6C. Bar, 2 mm.
International Journal of Systematic and Evolutionary Microbiology 63 Pseudonocardia antitumoralis sp. nov.
Table 2. Physiological characteristics of strain SCSIO 01299T and its closest phylogenetic neighbours in the genus Pseudonocardia Strains: 1, Pseudonocardia antitumoralis sp. nov. SCSIO 01299T; 2, P. ammonioxydans CGMCC 4.1877T; 3, P. kongjuensis DSM 44525T; 4, P.
autotrophica BCRC 12444T; 5, P. endophytica YIM 56035T; All data were taken from this study under the same culture conditions. All strains utilizearabinose, cellobiose*, D-galactose, glycerol, glucose, maltose and sodium oxalate as sole carbon sources, and are susceptible to chloramphenicol(30), ciprofloxacin (5), gentamicin (10), netilmicin (30), tobramycin (10) and vancomycin (30). +, Positive; 2, negative.
Diffusible pigment Milk peptonization Nitrate reduction Utilization of urea Sole carbon sources Antibiotic susceptibility (mg per disc) Erythromycin (15) Penicillin G (10) Sulfamethoxazole (24) Tetracycline (30) *For columns 2 and 5, not consistent with the original data.
DNot consistent with the original data.
X.-P. Tian and others Pseudonocardia zijingensis 6330 T (AF325725) Pseudonocardia adelaidensis EUM 221T (FJ805427) Pseudonocardia petroleophila ATCC 15777 T (X80596) Pseudonocardia xinjiangensis AS 4.1538 T (AF325728) Pseudonocardia yunnanensis IFO 15681T (D85472) Pseudonocardia aurantiaca DSM 44773 T (FR749916) Pseudonocardia rhizophila YIM 67013T (GU322368) Pseudonocardia thermophila IMSNU 20112T (AJ252830) Pseudonocardia chloroethenivorans SL-1T (AF454510) Pseudonocardia saturnea IMSNU 20052T (AJ252829) Pseudonocardia babensis VN05A0561T (AB514449) Pseudonocardia asaccharolytica DSM44247T (Y08536) Pseudonocardia mongoliensis MN08-A0270T (AB521671) Pseudonocardia ailaonensis YIM45505T (DQ344632) Pseudonocardia oroxyli D10 T (DQ343154) Pseudonocardia halophobica DSM 43089 T (Y08534) Pseudonocardia tetrahydrofuranoxydans K1T (AJ249200) Pseudonocardia benzenivorans B5 T (AJ556156) Pseudonocardia sulfidoxydans DSM 44248 T (Y08537) Pseudonocardia spinosispora LM 141T (AJ249206) Pseudonocardia eucalypti EUM 374 T (FJ805426) GMKU095 T (EU921261) Pseudonocardia carboxydivorans Y8 T (EF114314) Pseudonocardia antarctica DVS 5a1T (AJ576010) 99 Pseudonocardia alni IMSNU 20049T (AJ252823) Pseudonocardia tropica YIM 61452T (GQ906587) Pseudonocardia parietis 04-St-002T (FM863703) Pseudonocardia nitrificans IFAM 379 T (X55609) Pseudonocardia antitumoralis SCSIO 01299TP (JN204514)
Pseudonocardia ammonioxydans H9T (AY500143) Pseudonocardia endophytica YIM 56035T (DQ887489) Pseudonocardia kongjuensis LM 157 T (AJ252833) Pseudonocardia autotrophica IMSNU 20050 T (AJ252824) IMSNU 20111T (AJ252825) Actinophytocola burenkhanensis MN08-A0203T (AB535095) Actinokineospora riparia NRRL B-16432 T (AF114802) Allokutzneria albata DSM 44149 T (AJ512462) Fig. 2. Neighbour-method phylogenetic tree based on 16S rRNA gene sequences, showing the position of strain SCSIO01299T in the genus Pseudonocardia. Bootstrap values (.50 %) based on 1000 resamplings are shown at branch nodes.
Filled circles indicate that the corresponding nodes were also recovered in trees generated with the minimum-evolution andmaximum-likelihood methods. Bar, 0.5 % sequence divergence.
complete deletion gaps and minimum-evolution, max- pH 7.0–8.0, at 20–30 uC and with 3–8 % (w/v) NaCl. The imum-likelihood and maximum-parsimony methods, and isolate could use most of the sole carbon sources tested in confidence analysis was undertaken using 1000 bootstrap this study. The detailed physiological characteristics are replicates DNA–DNA hybridization was done according to the optical renaturation method An almost-complete 16S rRNA gene sequence (1435 bp) of ) using a UV-VIS spectropho- strain SCSIO 01299T was obtained. BLAST search results in tometer (model UV1601; Shimadzu).
EzTaxon-e showed that strain SCSIO 01299T had the highest Strain SCSIO 01299T had abundant white aerial mycelia, sequence similarities with Pseudonocardia ammonioxydans spores and yellowish white to moderate yellowish brown H9T (98.5 %) P. kongjuensis LM 157T substrate hyphae. The isolate grew well on ISP 2, 3, 4 and 5 (98.3 %) P. autotrophica IMSNU 20050T and Czapek's agar at 28 uC and grew weakly on potato (97.8 %) P. nitrificans IFAM 379T agar, but did not grow on nutrient agar. Dark orange (97.7 %) (), P. tropica YIM 61452T yellow pigment was produced on Czapek's agar. The (97.7 %) (P. endophytica YIM 56035T detailed cultural characteristics of this strain and its closest (97.6 %) and P. compacta IMSNU 20111T neighbours are listed in The organism formed an (97.6 %) (The neighbour-joining extensively branched substrate mycelia and aerial hyphae phylogenetic tree (showed that the isolate formed that differentiated into long spore chains. Spores were rod- a distinct branch that was most closely related to P. ammo- like with smooth surfaces (0.4–0.5 mm wide and 1.2– nioxydans H9T and more loosely related to P. kongjuensis LM 2.6 mm long) Cells of strain SCSIO 01299T were 157T, P. autotrophica IMSNU 20050T, and P. endophytica aerobic and Gram-positive. Good growth occurred at YIM 56035T, with high bootstrap support. These findings International Journal of Systematic and Evolutionary Microbiology 63 Pseudonocardia antitumoralis sp. nov.
Table 3. Chemotaxonomic characteristics of strain SCSIO 01299T and its closest phylogenetic neighbours in the genusPseudonocardia Strains: 1, Pseudonocardia antitumoralis sp. nov. SCSIO 01299T; 2, P. ammonioxydans CGMCC 4.1877T; 3, P. kongjuensis DSM 44525T; 4, P.
autotrophica BCRC 12444T; 5, P. endophytica YIM 56035T; Data were taken from this study unless otherwise stated. All strains contain Araand Gal as the diagnostic cellular sugars. DAP, Diaminopimelic acid; DPG, diphosphatidylglycerol; PC, phosphatidylcholine; PE,phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PIM, phosphatidylinositol mannosides; PL, unknown phospholipid;PME, phosphatidylmethylethanolamine; ND, not detected.
anteiso-C17 : 1v9c C17 : 0 10-methyl Amino acids/peptides in meso-DAP, Asp, Gly, meso-DAP, Asp, Gly, DPG, PC, PE, PMEc *Data were obtained from cells incubated in TSB with shaking at 28 uC for 5 days. Fatty acids representing ,0.5 % of the total in all taxa are notshown. Summed features represent two or three fatty acids that cannot be separated by the Microbial Identification System. Summed feature 3consisted of C16 : 1v7c and/or C16 : 1v6c. Summed feature 9 consisted of iso-C17 : 1v9c and/or C16 : 0 10-methyl.
DPhospholipid data were taken from: a, b, c, were supported with the minimum-evolution and max- genus Pseudonocardia. These cumulative data confirmed imum-likelihood analyses ) and the maximum- that the isolate should be assigned to the genus parsimony analysis (Fig. S1, available in IJSEM Online).
The chemotaxonomic data clearly showed the The cultural and micromorphological characteristics in differences between strain SCSIO 01299T and its four and including dark orange yellow soluble closest phylogenetic neighbours. The fatty acid composi- pigment on Czapek's agar, no growth on nutrient agar, tion of strain SCSIO 01299T contained a small amount of show the differences between strain SCSIO 01299T and its iso-C11 : 0 3-OH (1.03 %) and moderate amounts of four closest neighbours. As shown in the isolate anteiso-C17 : 0 (10.21 %) and summed feature 9 (compris- could use inositol, L-rhamnose, sodium oxalate and ing iso-C17 : 1v9c and/or C16 : 0 10-methyl; 10.85 %). The sodium citrate easily as sole carbon source for growth, polar lipids (Fig. S2) were diphosphatidylglycerol, phos- but not fucose, D-fructose or D-xylose. Strain SCSIO 01299T was susceptible to lincomycin and ampicillin, but not to erythromycin, rifampicin, sulfamethoxazole or nositol and three unknown phospholipids. There were no neomycin. The isolate could use cellulose for growth, was phosphatidylinositolmannosides present in the isolate. The positive for hydrolysis of starch and Tween 60 and milk type IV cell-wall composition of meso-diaminopimelic peptonization and was negative for utilization of urea, acid, arabinose and galactose production of H2S and hydrolysis of gelatin. Moreover, the predominant menaquinone MK-8(H4), and the strain SCSIO 01299T could grow at 4 uC and with up to major fatty acid iso-C16 : 0 were also consistent with the 15 % (w/v) NaCl. In conclusion, all of the above data could X.-P. Tian and others distinguish strain SCSIO 01299T easily from its closest The type strain, SCSIO 01299T (5DSM 45322T 5CCTCC relative, P. ammonioxydans CGMCC 4.1877T, and other M 2011255T), was isolated from a deep-sea sediment members of the genus Pseudonocardia.
sampled at a depth of 3258 m in the northern South ChinaSea. The G+C content of the genomic DNA is 73.2 mol%.
DNA–DNA hybridization showed that the isolate exhibited The type strain produces the previously synthesized low DNA–DNA relatedness with its closest phylogenetic antitumour agent deoxynyboquinone and its novel deri- neighbours (13 % with P. ammonioxydans CGMCC 4.1877T, vatives, pseudonocardians A–C.
25 % with P. autotrophica BCRC12444T and 18 % with P.
kongjuensis DSM 44525T), values which are below the 70 %cut-off point recommended for the delineation of genomic species (). It has been reported that,although many species in the genus Pseudonocardia share This research was supported by the National Basic Research Programof China (2010CB833801), the Knowledge Innovation Program of 98.5–99.6 % 16S rRNA gene sequence similarity, DNA– Chinese Academy of Sciences (KSCX2-YW-G-065, KSCX2-EW-G-12, DNA relatedness between them are below the 70 % cut-off KZCX2-YW-JC202), the National Natural Science Foundation of point ; These findings and China (40906075, 40906076, 41006089), the Natural Science Funds of results confirm that strain SCSIO 01299T represents a South China Sea Institute of Oceanology for Young Scholar genetically distinct species.
(SQ200901) and South China Sea Open Cruise by R/V Shiyan 3,South China Sea Institute of Oceanology, CAS.
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