Mrakia psychrophila sp. nov., a new species isolated from Antarctic soil

Mrakia psychrophila sp. nov., a new species isolated from Antarctic soil 1 first-author †E-mail:xinmingxiu@bnu.edu.cn †E-mail:xinmingxiu@bnu.edu.cn xinmingxiu@bnu.edu.cn 2 College of Life Sciences, Beijing Normal University, Beijing 100875, China College of Life Sciences, Beijing Normal University, Beijing 100875, China Institute of Microbiology, Academia Sinica, Beijing 100080, China Institute of Microbiology, Academia Sinica, Beijing 100080, China The yeast strain (Y18) was isolated from a soil sample collected from Fildes Peninsula, Antarctica. The strain is a psychrophilic yeast with optimum and maximum growth temperatures of 10 °C and 18 °C, respectively. Teliospores were formed after 7 d on malt agar, when the germination of teliospores was observed. Both inositol and D-glucuronate were assimilated. Positive results of the DBB (diazonium blue B) color reaction, urease test, and starch formation were observed. The major CoQ is Q 8 . All results indicated that Y18 belongs to the genes of Mrakia . The 18 S rDNA sequence analyses showed that Y18 is closely related to Mrakia frigida . DNA-DNA relatedness study, and some biochemistry characteristics indicated that Y18 represents a new species for which Mrakia psychrophila sp. nov. is proposed. Mrakia psychrophila Psychrophilic yeast 18 S rDNA sequence Teliospores INTRODUCTION 1 Psychrophiles are more likely to be found in permanently cold environments such as polar region, marine environment, and deep water (Sabri et al., 2001 2001 ; D′Amico et al., 2006 2006 ). In these environments, psychrophilic and psychrotrophic microorganisms are believed to play key roles in the biodegradation of organic matter and the cycling of essential nutrients (Welander, 2005 2005 ; Lambo and Patel, 2006 2006 ). Psychrophilic yeasts are very important due to their physiological adaptation and contributions to soil fertility, and they also have potential application in biotechnology. Ruberto et al.( 2005 2005 ) isolated some Rhodococcus strains having the ability to degrade hydrocarbon at low temperature. Cold active enzymes from psychrophilic yeasts can be applied to the food industry, for clarification of fruit juice and other fields at low temperature. Scorzetti et al.( 2000 2000 ) isolated a Cryptococcus adeliensis sp. nov. from Terre Adelie, Antarctica, which produced a cold-active xylanase. Nakagawa et al.( 2004 2004 ) isolated some psychrophilic yeast strains of Cryptococcus cylindricus and Mrakia frigida from soil of Abashiri. The isolated strains can grow on pectin as the only carbon at below 5 °C, and showed the activities of several cold-active pectinolytic enzymes. Cold active lipase A and lipase B from Candida antarctica have been expressed at Candida Antarctica and Escherichia coli , respectively, for the biotechnological application (Pfeffer et al., 2006 2006 ; Liu et al., 2006 2006 ). Kourkoutas et al.( 2002 2002 ) also reported the psychrophilic yeast can be used in low temperature fermentation. Leucosporidium was first introduced by Fell et al.( 1969 1969 ) as a heterobasidiomycetous yeast genus. Three types of CoQ system in this genus were found. The Q 10 system in L . capsuligenan and L . antaucticum , the Q 9 system in L . scottii , and the Q 8 system in L . frigidum , L . gelidum , L . nivale and L . stokesiin . The species with Q 8 system was later classified as the Mrakia species by Yamada and Komagata ( 1987 1987 ). Diaz and Fell ( 2000 2000 ) studied the internal transcribed spacer (ITS) and intergenic spacer (IGS) regions of rDNA from four psychrophilic Mrakia species. Babeva et al.( 2002 2002 ) reported a new species of Mrakia curviuscula from forest substrates collected in the central part of European Russia. There are 5 species in the Mrakia genus, with most of them being psychrophiles or psychrotrophs, and all of them isolated from low temperature environments. Recently, López-Archilla et al.( 2004 2004 ) isolated some Mrakia species from Tinto River. Margesin et al.( 2005 2005 ) studied the cold-active pectate lyases from psychrophilic Mrakia frigida . We report here the isolation and characteristics of a psychrophilic yeast strain Y18, isolated from soil samples near the Great Wall Station of China in Fildes Peninsula, Antarctica. MATERIALS AND METHODS 1 Yeast isolation 2 The soil samples were collected from Fildes Peninsula, Antarctica. Aliquots of the samples were suspended in malt extract and incubated on a rotary shaker at 0~5 °C for 3 d. Then the suspensions were diluted and spread on malt agar plates. After being incubated at 10 °C for 7 d, the colonies that appeared on the plates were replicated; one set was incubated at 10 °C and the other at 20 °C. The colonies that grew at 10 °C but did not at 20 °C were collected. Morphological, physiological and biochemical characterization 2 The morphological and physiological properties of the isolated strain were examined by using the methods of van der Walt and Yarrow ( 1984 1984 ). The studies of the life cycles were based on the methods of Fell et al.( 1969 1969 ). The range of growth temperatures and optimum temperature were determined by the method of van Uden ( 1984 1984 ). All tests were performed at 10 °C unless otherwise indicated. Ubiquinones were extracted and purified by using the method of Yamada and Kondo ( 1973 1973 ). The major ubiquinone was identified by high performance liquid chromatography (HPLC) as described by Nakase and Suzuki ( 1988 1988 ). 18 S rDNA sequencing and phylogenetic analysis 2 The 18 S rDNA region of strain Y18 was amplified by polymerase chain reaction (PCR) as described by Takashima and Nakase ( 1996 1996 ) using a pair of primers: P1 5′-TACCTGGTTGATCCTGCCAG-3′ and P2 5′-TAATGTCCTTCCGCAGGTCAC-3′. The PCR products were purified, ligated with pGEM-T vector, and then transformed into E . coli JM109. The recombinant plasmid containing the 18 S rDNA was isolated and the whole sequence of the 18 S rDNA fragment was determined. The 18 S rDNA sequence of the strain Y18 is deposited in GenBank with the accession number being AJ223490. The 18 S rDNA sequence of Y18 was aligned with that of other concerned yeast species by using Clustal W program version 1.7 (Thompson et al., 1994 1994 ). The phylogenetic tree was constructed with Treecon for Windows program version 1.2 (van de Peer and de Wachter, 1994 1994 ) using Neighbor-Joining analysis. Bootstrap sampling was performed based on 100 replicates. The following references of 18 S rDNA sequences were obtained from the nucleotide sequence databases (DDBJ, EMBL and GenBank): Filobasidiella neoformans (M55625), Tremella globipora (U00976), Tremella moriformis (U90977), Trichosporon cutaneum (X60182), Bullera crocea (D31648), Tremella foliacea (L22262), Filobasidium floriforme (D13469), Cryptococcus albidus (D31655), Mrakia psychrophila Y18 (AJ223490), Mrakia frigida (D12802), Phaffia rhodozyma (D31656), Leucosporidium lari-marini (D12805), Cystofilobasidium capitatum (D12801), Erythrobasidium hasegawianum (D12803), Rhodosporidium toruloides (D12806), Rhodotorula glutinis (X69853), Leucosporidium scottii (X53499), Sporobolomyces roseus (X60181), Sporidiobolus johnsonii (L22261). DNA base composition and DNA-DNA relatedness 2 DNA was isolated and purified by the methods of Zhu et al.( 1993 1993 ). DNA base composition was determined by the method of Ruan et al.( 1990 1990 ). For the DNA-DNA relatedness test, DNA probes were labeled with [α- 32 P]dCTP following the instruction of the nick translation kit (Boehringer Mannheim, Germany). Unlabelled DNA was dotted on the nitrocellulose membrane with the concentration of 25 μg/cm 2 . DNA hybridization was carried out according to the method of Molecular Cloning, a Laboratory Manual (2nd Ed.,1989). RESULTS 1 Phenotypical characteristics and generic assignment 2 Twelve psychrophilic yeast strains were isolated from soil samples of Fildes Peninsula, Antarctica. One strain designated as Y18 was selected for further studies for its low optimum temperature. Y18 is a psychrophilic yeast that can grow at 0 °C and at optimum and maximum growth temperatures of 10 °C and 18 °C, respectively. The DBB (diazonium blue B) color reaction, urease test, and starch formation were positive. The major ubiquinone system was Q 8 . The comparison of biochemical features between Y18 and other strains of Mrakia are shown in Table 1 1 . After one week on malt agar at 10 °C, hypha without clamp connection were produced. Teliospores formed after one week growth on malt agar. Pieces of agar with teliospores were dissected from the plate and placed in sterile distilled water and stored at 10 °C for 2 to 10 weeks. The teliospores were then streaked on agar plates (2%) and incubated at 10 °C. One-celled promycelium with terminal sporidia were observed after one week (Fig. 1 1 ). Species Y18 M . frigida M . gelida M . nivalis M . stokesii M . curviuscula mol% G+C 59 54.1 56.1 ND ND 49.3~50 Y18 homology (%) with 100 30 24 22 ND ND Phylogenetic analysis and DNA-DNA relatedness 2 The complete 18 S rDNA region of Y18 was sequenced and 1 806 bases were determined. Based on the alignment of the sequences compared, a phylogenetic tree was constructed using Neighbor-Joining method (Fig. 2 2 ). The strain Y18 was clustered together with Mrakia frigida . Subsequently, DNA-DNA homology values of Y18 with other species of Mrakia were examined. The data (Table 1 1 ) indicated that Y18 represents a new species clearly distinct from its phylogenetically closely related species. With the generic assignment consideration based on phenotypic characters as discussed above, and the finding that the strain is clustered together with Mrakia frigida , a new name of Mrakia psychrophila is proposed for Y18 with the specific epithet referring to the psychrophilic property of the species. Latin diagnosis of Mrakia psychrophila sp. nov. 2 In extracto malti 10 °C post 3 dies cellulae cylindricae, singulae, binae, aut catenatae, (3.0~4.5) μm×(7.5~15) μm, opt. temp. 10 °C, max. temp. 18 °C, crescentes ad 0 °C, generatae per gemmantem polarem. Post 30 dies pelliculae non efformatae sed cum sedimento ponderoso. In agaro malti post 7 dies colonia laevis, cremea in colori, mucosa et mollis in textura. Hyphae e cellulis singulis, uninucleatae, sine septis nodosis. Teliosporae intercalares aut terminales, sphericae aut ovoideae, (5~7) μm×(4~8) μm, incrementum ad (7~8) μm×(9~14) μm, singulae aut brevicatenatae. Fermentat Glucosum (lente), sucrosum (lente), cellobiosum (lente). Non fermentat galactosum, maltosum, melibiosum, lactosum, melezitosum, raffinosum, inulinum. Assimilat galactosum, sorbosum (exigue), D-glucosaminum, D-ribosum (exigue), D-xylosum, L-arabinosum, D-arabinosum, rhamnosum, maltosum, trehalosum, α-methyl-D-glucosidum, salicinum, arbutinum, melibiosum, lactosum, raffinosum, melezitosum, inulinum, amylum solubile, glycerolum, ergthritolum, ribitolum, xylitolum, D-glucitolum, mannitolum, inositolum, D-glucono-1,5-lactonum, D-gluconatum, D-glucuronatum (exigue), lacticum, succinicum, citricum, methanolum (exigue), ethanolum, kalium nitricum, ethylaminum, L-lysinum, creatinum, creatininum. Non assimilat erythritolum, DL-lacticum, citricum, sodiinitrosum. Crescit sine vitamines; amylum formatur; findendum arbutini absens; incrementum in 50% agaro glucosi extracti fermenti positivum; urea finduntur; Diazonium Blue B respondet; cum cycloheximido 0.01% crescit; G+C, 59.0 mol%; ubiquinonum majus, Q 8 . Holotypus: AS 2.1971 (originaliter ut Y18) conseratur in collectionibus culturarum quas ‘China General Microbiological Culture Collection Center’, Instituti Microbiologici Academiae Sinicae, Beijing sustentat. Description of Mrakia psychrophila sp. nov. 2 Growth in malt extract: After 3 d at 10 °C, the cells were cylindrical, (3~4.5) μm×(7.5~15) μm, (Fig. 1a 1a ), reproducing by polar budding. After one month there was no formation of pellicle but thick sediment. Growth on malt agar: after one week, the colony was smooth, cream-colored, mucousy and soft in texture. The colony border was fringed with mycelia. Mycelia without clamp connections were present (Fig. 1b 1b ), and the telispores were formed terminally on the mycelia (Fig. 1c 1c ). Germination of teliospores was observed and one-celled promycelium with terminal sporidia was observed (Fig. 1d 1d ). The fermentation and assimilation of carbon and nitrogen source are shown in Table 2 2 . Note: “−”, “+”, “D”, “s”, “V”, “nd” stand for “negative”, “positive”, “delayed positive”, “slow growth”, “variable growth”, and “not determined”, respectively 2 Characteristic 6 Reaction of strains Y18 M . frigida M . gelida M . nivalis M . stokisii M . curviuscula Isolated site Antarctic soil Antarctic soil Antarctic soil Antarctic soil Antarctic soil Forest of Russia Maximum temperature (°C) 18 19 20 20 20 25 7 Fermentation of Sucrose + D D D V nd Cellobiose D − − − − nd 7 Assimilation of Sorbose + V + V V − D-Ribose + − V V V s D-Arabinose + D V D − V Maltose + − + − + + Methyl + − V − − nd Arbutin − + + + + nd Melibiose + − + V − − Raffinose + + + + + − Melezitose + − + − + + Inulin + − − − − − Starch + − + − + V Glycerol + − V V V − myo-Inositol + D V V V − DL-Lactate − V V D V s Citrate − + V + + + Methanol + − − − − nd NaNO 2 − + + + + nd Ethylamine + − − − − nd L-Lysine + V + − + nd Creatine + − − − − nd Creatinine + − − − − nd 7 Others Without vitamin + − − − − − With 50% glucose (w/v) + − − − − nd Maximum growth temperature: 18 °C; growth in vitamin-free medium: positive; starch formation: positive; splitting of arbutin: negative; growth on 50% (w/w) glucose yeast extract agar: positive; urease: positive; DBB reaction: positive; growth in the presence of 0.01% cycloheximide: positive; G+C content of the nuclear DNA: 59.0 mol%; major ubiquinone: Q 8 . The type strain Y18 was deposited in the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, Beijing, with the strain number AS 2.1971. DISCUSSION 1 The results of morphology, biochemistry and physiology studies of Y18 support that Y18 belongs to the genus of Mrakia . The phylogenic tree showed Y18 classified into one group with Mrakia frigida , and the 18 S rDNA sequence analysis also showed high homology (98.93%) between Y18 and Mrakia frigida . There are some distinct differences between Y18 and other species of Mrakia as shown in Table 2 2 . Y18’s growth in 50% glucose-yeast extract agar, in presence of 0.01% cycloheximide, and without external vitamins were observed. Cellobioses, and sucrose were fermented by Y18. The optimum and maximum temperatures were low compared with other Mrakia species. DNA-DNA relatedness (Table 1 1 ) also supported that Y18 and M . frigida are not of the same species. 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Nucleic Acids Res , 1993 . 21 ( 22 ): 5279 - 5280 . 8255788 DOI: 10.1093/nar/21.22.5279 http://doi.org/10.1093/nar/21.22.5279 . Isolation of genomic DNAs from plants, fungi and bacteria using benzyl chloride We thank Prof. Zheng Ruyong, Institute of Microbiology, Chinese Academy of Sciences, for her advice and correction of Latin description on the manuscript. 10.1631/jzus.2007.B0260 Project (No. 30170001) supported by the National Natural Science Foundation of China 2006-10-19 2006-12-18 2022-01-05 Copyright © 2007, Journal of Zhejiang University Science 2007 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 04 08