View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Kyoto University Research Information Repository Stimulation of methanotrophic growth in cocultures by Title cobalamin excreted by rhizobia. Author(s) Iguchi, Hiroyuki; Yurimoto, Hiroya; Sakai, Yasuyoshi Applied and environmental microbiology (2011), 77(24): 8509- Citation 8515 Issue Date 2011-12 URL http://hdl.handle.net/2433/152321 Right © 2011, American Society for Microbiology. Type Journal Article Textversion author Kyoto University 1 Stimulation of methanotrophic growth in co-cultures by 2 cobalamin excreted by rhizobia 3 4 Hiroyuki Iguchi,1 Hiroya Yurimoto,1 and Yasuyoshi Sakai1,2* 5 6 Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto 7 University, Kyoto,1 and Research Unit for Physiological Chemistry, the 8 Center for the Promotion of Interdisciplinary Education and Research, 9 Kyoto,2 Japan 10 11 Corresponding author: Yasuyoshi Sakai, Ph.D. Professor 12 Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto 13 University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto 606-8502, Japan. 14 Tel: +81 75 753 6385. Fax: +81 75 753 6454 15 E-mail: [email protected] 16 17 Running title: Cobalamin stimulates methanotrophic growth 18 19 1 20 ABSTRACT 21 Methanotrophs play a key role in the global carbon cycle, in which they 22 affect methane emissions and help to sustain diverse microbial communities 23 through the conversion of methane to organic compounds. To investigate the 24 microbial interactions that caused positive effects on the methanotroph, co-cultures 25 were constructed using Methylovulum miyakonense HT12 and each of nine 26 non-methanotrophic bacteria, which were isolated from a methane-utilizing 27 microbial consortium culture established from forest soil. Three rhizobial strains 28 were found to strongly stimulate the growth and methane oxidation of M. 29 miyakonense HT12 in co-cultures. We purified the stimulating factor produced by 30 Rhizobium sp. Rb122, and identified it as cobalamin. Growth stimulation by 31 cobalamin was also observed for three other -proteobacterial methanotrophs. These 32 results suggest that microbial interactions through cobalamin play an important 33 role in methane oxidation in various ecosystems. 34 2 35 INTRODUCTION 36 Methane is the second most important greenhouse gas, and mitigating 37 emissions of methane has become a major global concern (17). Aerobic 38 methanotrophs are the major terrestrial methane sink and are widespread in a 39 large variety of ecosystems (26). They belong to the -Proteobacteria (type I), 40 -Proteobacteria (type II) and Verrucomicrobia (26). Methanotrophs utilize 41 methane as a single source of carbon and energy, but only some methanotrophic 42 strains in the class -Proteobacteria can assimilate substrates with C-C bonds (8). 43 Mutual interactions that occur between methanotrophs and other 44 organisms, ranging from microbes to plants and animals, may affect the global 45 methane cycle in various ways. Stable isotope probing (SIP) experiments revealed 46 that methane-derived carbon was incorporated into methylotrophic or heterotrophic 47 bacteria when they were incubated with methanotrophs (5, 14, 20, 22-23), 48 indicating an important role of methanotrophs in supplying nutrients in the forms 49 of carbon sources to other non-methanotrophic organisms. At deep-sea 50 hydrothermal vents and cold seeps, invertebrates form symbiotic associations with 51 -proteobacterial methanotrophs living in their tissues (21, 26). Some invertebrates 52 can derive most of their carbon nutrition from methane, indeed acquiring it from 53 methane-derived metabolites of methanotrophs or by digestion of methanotrophs. 54 Inversely, the hosts provide methanotrophs with simultaneous access to methane 3 55 and oxygen by positioning themselves appropriately and also by providing a stable 56 environment. In peat bogs, Sphagnum mosses associate with -proteobacterial 57 methanotrophs and utilize carbon dioxide that is generated from methane by 58 methanotrophic symbionts (18, 24). Although previous studies have demonstrated 59 that methanotrophs serve as food suppliers for other organisms, less attention has 60 been paid to the specific benefits that methanotrophs acquire from such 61 interactions. 62 Previously, we established a microbial consortium from forest soil utilizing 63 methane as the single carbon and energy source, from which we isolated a new 64 obligate methanotroph Methylovulum miyakonense HT12 (15). The aim of the 65 present study is to clarify the beneficial factors for methanotrophic growth and 66 methane oxidation provided by other bacteria in the methane-grown microbial 67 consortium to further understand interactions between methanotrophs and 68 microorganisms. 4 69 MATERIALS AND METHODS 70 Methanotrophic strains and growth conditions. M. miyakonense HT12 71 was a lab stock (15). Methylococcus capsulatus Bath, Methylobacter luteus, 72 Methylomonas methanica S1 and Methylosinus trichosporium OB3b were obtained 73 from the culture collection of NCIMB (No. 11132, No. 11914, No. 11130 and No. 74 11131, respectively). Details of the isolation of methanotrophic strains OS501, R4F, 75 B3R, SH31p and SS2C will be described elsewhere: Iguchi H, Sato I, Sakakibara M, 76 Yurimoto H and Sakai Y, in preparation. The methanotrophic strains were grown in 77 nitrate mineral salts (NMS) medium (28) under a 20 : 80 methane to air atmosphere 78 at 28ºC with shaking in 25 ml vials capped with butyl-rubber stoppers. In order to 79 compensate for the slow growth, M. miyakonense HT12, strain OS501 and strain 80 R4F were subcultured in medium supplemented with Bacto tryptone (Becton, 81 Dickinson and Company) at 0.01% (w/v). 82 Isolation of bacterial strains grown in the consortium culture. The 83 methane-enriched culture of forest soil (15) was subcultured every month over two 84 years. The culture was serially diluted and spread onto agar plates: the media used 85 were modified Luria-Bertani (LB) broth (0.2% tryptone, 0.1% yeast extract, 0.1% 86 sodium chloride, 0.1% glucose), 10-fold diluted Tryptone Soya Broth (OXOID, 87 Cambridge, UK), 5-fold diluted Nutrient Broth (Becton, Dickinson and Company) 88 and NMS medium with 0.1% methanol. A single colony grown on the plate at 28ºC 5 89 was isolated. The 16S rRNA gene was amplified by PCR using the 27f-1492r primer 90 set (27) and sequenced. A similarity search for the nucleotide sequence of the 16S 91 rRNA gene of the isolates was carried out using the BLAST program 92 Mixed culture experiments. The cells of the heterotrophic isolates were 93 prepared from liquid cultures grown in modified LB broth. Because of the inability 94 to grow in this medium, strain Rb122 was grown in TY medium (0.5% tryptone, 95 0.3% yeast extract, 0.083% calcium chloride). Methanotroph cells were prepared 96 from liquid cultures in NMS medium with methane. Cells of the methanotroph and 97 the heterotroph were washed with water, and then mixed to adjust the OD600 ratio 98 to 10 : 1 (OD600 values, 0.0025 and 0.00025, respectively), and cultivated in NMS 99 medium with methane. For the second generation culture, the cells were harvested 100 by centrifugation from the 8 day-old mixed culture, washed with water and 101 inoculated into fresh NMS medium at an OD600 value of 0.0025. Triplicate samples 102 were prepared for each culture and for each time point, because after the methane 103 concentration was measured the vial was opened to measure the OD600. 104 Methane concentrations were determined using a Shimadzu GC-14B gas 105 chromatograph (Shimadzu, Kyoto, Japan) equipped with a flame ionization detector 106 and Porapak Q column (Shinwa chemical industries, Kyoto, Japan). Nitrogen gas 107 was used as the carrier. Analytical temperatures of the oven, injector and detector 108 were 100, 120 and 225ºC, respectively. 6 109 Quantitative PCR (qPCR). qPCR was carried out using a LightCycler 110 system (Roche Diagnostics, Tokyo, Japan) and SYBR Premix ExTaq (Takara Bio, 111 Shiga, Japan) according to the manufacturer’s instructions. Genomic DNA, which 112 was extracted from the culture by a method using SDS and proteinase K as 113 described previously (16), was used as template. Primers specific for the 16S rRNA 114 gene of each strain were designed. The primer sets were ht-fw 115 (TGGCCCCAATTATGGGGTAA) and ht-re (AGGGATCTCTGCCGAATCCA) for M. 116 miyakonense HT12, and rb-fw (GTCGGGCAGTTGACTGTTCG) and rb-re 117 (TACCGTCTCCGGTAACCGCGA) for strain Rb122. A standard curve for copy 118 number calculation was generated with plasmid DNA (pGEM-T Easy [Promega, 119 Madison, WI] harboring the 16S rRNA gene). 120 Preparation of spent medium and cell extracts. The pure culture of M. 121 miyakonense HT12, and the co-culture of M. miyakonense HT12 and strain Rb122 122 were grown in NMS medium with methane. Strain Rb122 was grown in Rhizobium 123 minimal medium (RMM) with glucose (13). Bacterial cells were removed from the 124 culture by centrifugation, and the spent medium was filter-sterilized using a 0.45 125 m filter. Until use, the samples were stored at -80 ºC. 126 For quantification, cobalamins were extracted from the lyophilized culture 127 or the harvested cells with methanol twice by incubating at 80ºC. The extracts were 128 lyophilized, dissolved in water and filter-sterilized using a 0.45 m filter. 7 129 Stimulatory activity assay on methanotrophic growth (the HT12 assay). 130 M. miyakonense HT12 was grown on methane in NMS medium containing a test 131 sample. The stimulatory activity of the sample was evaluated based on the OD600 132 value after cultivation for 48-72 hr. We confirmed that the addition of NMS medium, 133 RMM or methanol extracts of the media did not stimulate growth. For 134 quantification of the stimulatory activity, a calibration curve was established using 135 a cyanocobalamin standard (Wako Pure Chemical Industries, Osaka, Japan). 136 Bioassay of cobalamin (the Lactobacillus assay). Cobalamin was 137 quantified by the growth of Lactobacillus delbrueckii NBRC 3073 on B12 Assay 138 Medium (Becton, Dickinson and Company) according to the Difco & BBL Manual.