Short Communication Detection of Betaproteobacteria Inside the Mycelium of the Fungus Mortierella Elongata
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Microbes Environ. Vol. 25, No. 4, 321–324, 2010 http://wwwsoc.nii.ac.jp/jsme2/ doi:10.1264/jsme2.ME10134 Short Communication Detection of Betaproteobacteria inside the Mycelium of the Fungus Mortierella elongata YOSHINORI SATO1, KAZUHIKO NARISAWA1,2, KAZUTO TSURUTA2, MASAFUMI UMEZU2, TOMOYASU NISHIZAWA2, KENJI TANAKA3, KAORU YAMAGUCHI3, MASAKAZU KOMATSUZAKI1,2, and HIROYUKI OHTA1,2* 1Institute for Global Change Adaptation Science, Ibaraki University, 2–1–1 Bunkyo, Mito, Ibaraki 310–8512, Japan; 2Ibaraki University College of Agriculture, 3–21–1 Chuo, Ami-machi, Ibaraki 300–0393, Japan; and 3Biological Resource Center (NBRC), Department of Biotechnology, National Institute of Technology and Evaluation, 2–5–8 Kazusakamatari, Kisarazu, Chiba 292–0818, Japan (Received May 25, 2010—Accepted September 4, 2010—Published Online September 25, 2010) Microscopic and molecular analyses showed the presence of endobacteria inside the mycelia of four out of twelve nitrous oxide (N2O)-producing fungal isolates identified as Mortierella elongata. The 16S rRNA gene was successfully amplified with DNA extracted directly from the endobacterium-containing fungal strains and all sequences were related to that of Candidatus Glomeribacter gigasporarum in the family Burkholderiaceae. Bacterial endotoxin was detected in the endobacterium-positive fungal strains but only trace levels were found in endobacterium-negative strains. No significant relationship was found between the fungal N2O-producing activity and the presence of endo- bacteria. Key words: endobacterium, N2O production, Mortierella elongata, soil fungus The intracellular distribution of bacteria, or more spe- Tsuruta, K. Narisawa, M. Komatsuzaki, N. Kaneko, H. Ohta, cifically bacterial endosymbiosis, is known for numerous submitted for publication). Comparisons were made with eukaryotic organisms including ciliate protozoa (25), various herbarium materials, Mortierella elongata Linnemann insects (11), a frenulata (marine invertebrate) (12), and well- NBRC 8570 and M. elongata MAFF 425591, obtained known legume roots. Concerning the association of bacteria from the National Institute of Technology and Evaluation with fungi, bacterial endosymbiosis has been reported in (NITE Biological Resource Center, Chiba, Japan), and some strains of Glomeromycota [e.g. Geosiphon pyriform National Institute of Agrobiological Sciences (NIAS Gene- (22), and Gigaspora and Scutellospora species (4, 6, 7, 15)], bank, Ibaraki, Japan), respectively. Ascomycota [e.g. Tuber borchii (1)], Basidiomycota [e.g. The morphology of M. elongata strains was observed Laccaria bicolor; (3)] and Zygomycota [e.g. Rhizopus by fluorescence microscope. Briefly, the mycelia of a species (10, 18)]. In our recent study, nitrous oxide (N2O)- culture grown for 3 days at 23°C on half-strength cornmeal- producing fungal strains were isolated from upland soils and malt-yeast agar (CMMY) {grams per liter: cornmeal agar the intramycelial localization of bacterium (endobacterium) [Becton-Dickinson (BD), Franklin Lakes, NJ, USA], 8.5; in the fungal isolates was found (Sato, Y., et al. 2008. malt extract (BD), 10; yeast extract (BD), 2; agar (BD), 7.5} Abstracts for the 12th International Symposium on Microbial was mounted on a polycarbonate membrane filter (black Ecology, Cairns, Australia). The purpose of this study is to filter with 0.2 µm pore size, Advantec, Tokyo, Japan) and examine further the presence of endobacteria using fluores- stained for 15 min with a LIVE/DEAD BacLight Bacterial cence and electron microscopy, the amount of endotoxin, the Viability Kit (Molecular Probes, Eugene, OR, USA) (8) that 16S rRNA gene, and the relationship between the presence is specific for bacteria and can distinguish between live and of endobacteria and fungal N2O-producing activity. dead cells. The filter was rinsed with filter-sterilized (0.2 µm Ten fungal strains used in this study were isolated from pore size, Advantec) distilled water and placed on a micro- no-tilled, low-nitrogen input cropland soils at the Field scope slide. Preparations were observed under blue and Science Center, Ibaraki University College of Agriculture, green light with a fluorescence microscope (BX51, Olympus, located in the Kanto plains of Japan and were identified as Tokyo, Japan) equipped with a charge-coupled device phylum Zygomycota, Mortierella elongata from their (CCD) camera (DP-50, Olympus). Green-fluorescent bacteria- morphological characteristics and their 18S-26S/28S internal like organelles (BLOs) were detected inside the mycelia transcribed spacer sequence (ITS). All of the M. elongata of strains FMR23-1 (Fig. 1A), FMR23-6, FMR23-9, and strains were examined for activity to produce N2O and FMR13-2. Further observation showed that the BLOs existed found to have low activity levels during growth in cultures also inside the fungal spores of all endobacterium-containing containing 10 mM nitrite but no activity in cultures with strains (data not shown). No BLOs were found in the 10 mM nitrate (Y. Sato, T. Nishizawa, M. Umezu, K. reference strains M. elongata NBRC 8570 and MAFF 425591. To our knowledge, there is no report that the LIVE/ * Corresponding author. E-mail: [email protected]; DEAD BacLight Bacterial Viability Kit is applicable to the Tel: +81–29–888–8684; Fax: +81–29–888–8525. mycelium of M. elongata. Therefore, we do not make any 322 SATO et al. and some BLOs were attached to secondary hyphae of M. elongata FMR23-6 (Fig. 2A). A magnified image of endo- bacteria in the fungal cytoplasm showed that they possessed a double-layered cell envelope and cytoplasm rich in ribo- somes (Fig. 2B). These microscopic results suggest the presence of endobacteria, probably double-layered Gram- negative bacteria, inside the mycelia of the M. elongata strains. To characterize the endobacteria, PCR amplification was carried out in a reaction mixture containing 100 ng of template DNA extracted from fungal mycelia using the ISOPLANT DNA extraction kit (Nippon Gene, Tokyo, Fig. 1. Fluorescence microscopic observation of the fungal mycelia with the Live/Dead Baclight kit. A, green fluorescent bacterium-like Japan) according to the manufacturer’s instructions. The prim- organelles were found in the mycelia of Mortierella elongata FMR23- ers for the bacterial 16S rRNA gene were 10F primer (5'- 1; B, no bacterium-like organelle was detected in the mycelia of AGTTTGATATCCTGGCTCAG-3', corresponding to posi- µ FMR23-3. Bars, 20 m. tions 10–27 of the E. coli 16S rRNA gene) and 1541R primer (5'-AAGGAGGTGATCCAGCCG-3', positions 1524–1541). presumption as to the viability of the fungal mycelium from The reaction profile was as follows: initial denaturation at the image presented in Fig. 1. 95°C for 5 min followed by 25 cycles of denaturation at To further characterize the intracellular distribution of 95°C for 30 s, annealing at 60°C for 1 min and extension at the bacteria, ultrathin sections of the mycelia were prepared 72°C for 1 min. Amplified fragments (approximately 1.5 kb) and examined with transmission electron microscopy. The were obtained from FMR23-1, FMR23-6, FMR23-9 and growing mycelium of M. elongata FMR23-6 grown for 3 FMR13-2, while no 16S rRNA gene fragment was amplified days at 23°C on CMMY was fixed with 3.5% glutaraldehyde from the other 10 strains using universal bacterial primers in 1/15 M potassium phosphate buffer (pH 7.0) for 1 h at (Table 1). room temperature. After further fixing with a 2% (w/v) To check the endobacterial diversity associated with the osmium tetraoxide (OsO4) solution for 1 h at room tem- single fungal strain, terminal restriction fragment length poly- perature, the sample was embedded in Spurr’s resin (24). morphism(T-RFLP)fingerprintingwasconductedasdescribed Ultrathin sections were obtained with a glass knife on an previously (21). Briefly, the PCR for T-RFLP fingerprinting ultramicrotome Ultracut UCT (Leica Microsystems, Wetzlar, (14) was performed using a 5'-end fluorescence (BODIPY Germany), stained with 0.5% (w/v) potassium permanganate FL)-labeled primer (Q-10F; E. coli positions 10–27) pur- (KMnO4) in a 1/15 M potassium phosphate buffer (pH 7.0) chased from J–Bio21 (Tsukuba, Japan) and 926r (E. coli for 1 min, and rinsed with a 0.05% (w/v) citric acid solution positions 907–926) under conditions of 2 min at 95°C, then for 10 s, then post-stained with lead citrate (20) for 5 min and 15 cycles of 95°C (30 s), 54°C (45 s), and 72°C (1.5 min). observed under a Hitachi H-7600 transmission electron The fluorescently labeled-PCR products were purified using microscope (Hitachi, Tokyo, Japan) at 100 kV. a Qiagen DNA purification Kit (Qiagen, Hilden, Germany) µ µ Transmission electron microscopy revealed that M. and eluted in a final volume of 30 L. Aliquots (5 L) of elongata strain FMR23-6 formed intrahyphal hyphae inside the amplified product were separately digested with HaeIII, the cytoplasm in the primary mycelium (Fig. 2A). This MspI, and RsaI (Takara Bio, Otsu, Japan) for 2 h according unique structure was already reported for Mortierella to the manufacturer’s instructions. The labeled fragments multidivaricata, in a subgenus, Gamsiella, of Mortierella (2). were purified with the Qiagen DNA purification kit and According to the description, the intrahyphal hyphae arise separated on the Applied Biosystems 3130xl DNA Sequencer not infrequently from the end of the living cell segment of the ageing aerial mycelium of M. multidivaricata (2). The Table 1. The PCR amplification and Limulus amebocyte lysate test BLOs were observed in the cytoplasm of primary hyphae for detecting endobacteria in the mycelia of Mortierella elongata strains. Et concentration Strain (accession number) PCR amplification of 16S rRNA gene* (µg Et g wet mycelia−1) FMR23-1 (AB542099) + 70 FMR23-2 (AB542100) −<0.1 FMR23-3 (AB542101) − 0.4 FMR23-6 (AB542104) + 240 FMR23-9 (AB542107) + 420 FMR23-10 (AB542108) −<0.1 FMR13-1 (AB542092) −<0.1 FMR13-2 (AB542093) + 250 FMR13-3 (AB542094) − 0.3 FMR13-5 (AB542096) −<0.1 Fig. 2. Transmission electron micrographs of endobacteria inside the MAFF 425591 (AB542112) −<0.1 fungal mycelia of Mortierella elongata FMR23-6.