Caloramator Coolhaasii Sp. Nov., a Glutamate- Degrading, Moderately Thermophilic Anaerobe
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International Journal of Systematic and Evolutionary Microbiology (2000), 50, 1155–1162 Printed in Great Britain Caloramator coolhaasii sp. nov., a glutamate- degrading, moderately thermophilic anaerobe Caroline M. Plugge,1 Erwin G. Zoetendal1,2 and Alfons J. M. Stams1 Author for correspondence: Caroline M. Plugge. Tel: j31 317 482105. Fax: j31 317 483829. e-mail: Caroline.Plugge!algemeen.micr.wau.nl 1 Laboratory of An obligately anaerobic, moderately thermophilic, glutamate-degrading Microbiology, Wageningen bacterium (strain ZT) was isolated from an enrichment culture obtained from University, H. van Suchtelenweg 4, anaerobic thermophilic granular sludge. The cells were rod-shaped to 6703 CT Wageningen, filamentous and showed no motility or spore formation. The cell wall had a The Netherlands Gram-positive structure, which was revealed by electron microscopy. Optimum 2 Wageningen Centre for growth of the strain was observed under neutrophilic conditions at 50–55 SC. Food Sciences, PO Box 557, The doubling time of strain ZT grown in rich medium was approximately 1 h at 6700 AL Wageningen, T The Netherlands optimal pH and temperature. Strain Z was able to grow on a variety of organic compounds. Most carbon sources were converted to acetate, CO2,H2, and T traces of propionate and lactate. Strain Z oxidized glutamate to acetate, CO2, M NH4 , traces of propionate and H2. The doubling time on this substrate was 16d. The strain fermented glutamate syntrophically in co-culture with Methanobacterium thermoautotrophicum Z-245T to the same products, but the co-culture had a fourfold higher growth rate. 16S rDNA sequence analysis revealed a relationship with Thermobrachium celere, Caloramator indicus and Caloramator proteoclasticus. The GMC content was 317 mol%. Based on its morphological, phylogenetic and physiological characteristics, it is proposed that strain ZT should be classified in the genus Caloramator as a new species, Caloramator coolhaasii. Keywords: Caloramator coolhaasii sp. nov., interspecies hydrogen transfer, glutamate degradation, methanogenic archaea, syntrophic consortia INTRODUCTION mate in co-culture with hydrogen-scavenging meth- anogens (Cheng et al., 1992; Tarlera, 1997; Baena et Proteins are important substrates in anaerobic di- al., 1999a; Nanninga & Gottschal, 1985; Stams & gestion (Barker, 1981; McInerney, 1989). Amino acids, Hansen, 1984). Some of these organisms are meso- the hydrolysis products of proteins, can serve as carbon philic, but Selenomonas acidaminovorans, recently and energy sources for many types of anaerobic renamed Thermanaerovibrio acidaminovorans (Baena bacteria. Utilization of amino acids has been used as a et al., 1999b), and Caloramator proteoclasticus are classification characteristic for clostridia (Mead, moderately thermophilic. These bacteria grow slowly 1971; Elsden & Hilton, 1979). Several anaerobic in pure culture, but more rapid growth is achieved bacteria have been described that can grow on gluta- when grown syntrophically with a partner organism mate. Most of them are members of the low-GjC which removes the hydrogen. These bacteria degrade − + Clostridium subphylum, which convert glutamate to one glutamate to two acetate, one HCO$, one NH% + − acetate, butyrate, CO#,NH% and traces of hydrogen and one H#, or to one propionate, two HCO$, one + (Buckel & Barker, 1974; Laanbroek et al., 1979). Also, NH% and two H#, where the ratio is dependent on the fermentative anaerobes are described that utilize gluta- hydrogen partial pressure. In this paper, the properties of strain ZT, a novel ................................................................................................................................................. organism isolated from anaerobic methanogenic Abbreviations: MPN, most probable number; TEM, transmission electron granular sludge, are described. This bacterium microscopy. degrades glutamate in pure culture, but much faster The GenBank accession number for the 16S rDNA sequence of strain ZT is growth is achieved in co-culture with hydrogeno- AF104215. trophic methanogens. 01148 # 2000 IUMS 1155 C. M. Plugge, E. G. Zoetendal and A. J. M. Stams (a) (c) (d) (b) ................................................................................................................................................................................................................................................................................................................. Fig. 1. (a, b) Phase-contrast micrographs of cells of strain ZT during growth on glutamate (a) and WC broth (b); bar, 10 µm. (c, d) Electron micrographs showing (c) the single-layered structure of the cell wall (bar, 0n1 µm; cm, cytoplasmic membrane; p, peptidoglycan layer) and (d) cell division (bar, 1 µm; sf, septum formation). Electron-dense structures are probably intracellular protein precipitates (d). 1156 International Journal of Systematic and Evolutionary Microbiology 50 Caloramator coolhaasii sp. nov. METHODS Philips EM400 TEM. Motility was determined using Gray’s T method for flagella staining (Gerhardt et al., 1981). For Organisms, cultivation and isolation procedures. Strain Z negative staining, cells were fixed for 1 h in 2n5% (v\v) was isolated from thermophilic methanogenic granular glutaraldehyde in 0n1 M sodium cacodylate buffer (pH 7n2). sludge converting glutamate as the sole source of carbon and A Formvar-coated grid (150 mesh) was placed on a drop of energy. Methanobacterium thermoautotrophicum Z-245T (l T T a dense cell suspension for 20 min. After rinsing with de- DSM 3720 ) and Thermobrachium celere DSM 8682 were ionized water, the cells were stained with 1% (w\v) uranyl obtained from the German Collection of Microorganisms acetate and the grid was air-dried. Cells were examined in a and Cell Cultures (DSMZ, Braunschweig, Germany). Calo- JEOL TEM. ramator proteoclasticus DSM 10124T was from our own culture collection. GjC content. The GjC content of the DNA was de- et al A bicarbonate-buffered anaerobic medium as described termined at the DSMZ by HPLC (Mesbah ., 1989). previously by Stams et al. (1993), supplemented with 0n02% SDS-PAGE analysis of whole-cell protein profiles. The strains yeast extract, was used for enrichment and cultivation of were grown at 55 mC in bicarbonate-buffered anaerobic strain ZT. Wilkens–Chalgren broth (WC broth; Oxoid; 16 g −" T medium supplemented with 0n2% yeast extract and 10 mM l ) was used for the isolation of strain Z . Direct dilution glucose. Samples from cells in the exponential phase (cor- series in liquid WC broth as well as agar plates supplied with responding to OD'!! values of approximately 0n25) were WC broth were used. All incubations were done at 55 mCin centrifuged for 5 min at 16000 g. The pellets were the dark, unless otherwise stated. For direct comparison of resuspended in 100 mM Tris\HCl buffer (pH 7n2). Protein strain ZT with Caloramator proteoclasticus and T. celere, −" samples were diluted in denaturing loading buffer and boiled all cultures were cultivated on: WC broth (16 g l ); for 15 min. Cell debris was removed by centrifugation bicarbonate-buffered anaerobic medium (Stams et al., 1993); (5 min at 16000 g) and samples were stored at 4 mC until bicarbonate-buffered anaerobic medium supplemented with electrophoresis. SDS-polyacrylamide running gels with 10% 0n02% yeast extract; bicarbonate-buffered anaerobic acrylamide and 4% acrylamide stacking gels were run in a medium supplemented with 0n2% yeast extract; and −" Bio-Rad Mini Protean II system at 20 mA for 2 h. Approxi- YTG medium. YTG medium contained (l ): Na#CO$, mately 10 µg protein was loaded in each lane. High- 5 3g;NaHPO .2H O, 0 356 g; KCl, 0 075 g; yeast extract n # % # n n molecular-weight markers for SDS-PAGE were from LKB- 5 g; tryptone, 10 g; cysteine.HCl, 0 2g;NaS.9H O, 0 2g; n # # n Pharmacia. The gels were stained with 0 1% (w\v) resazurin, 1 mg. Before use, the pH of the YTG medium was n Coomassie brilliant blue R250 in methanol\deionized adjusted to 10 by adding sterile 3 M NaOH. Glucose water\acetic acid (4:5:1, by vol.) overnight and destained in (10 mM) was used as the carbon source, except in the case of WC broth, where no additional carbon source was added. methanol\deionized water\acetic acid (4:5:1, by vol.) for 3h. Determination of growth parameters. The growth rate of T 16S rDNA sequence analysis. Genomic DNA was isolated strain Z on WC broth was determined by measuring the T increase in OD'!! in triplicate with time. The growth rate on from strain Z and the 16S rDNA was amplified using glutamate with and without yeast extract was determined by bacterial primers 8f and 1510r (Lane, 1991) under previously measurement of product formation with time. Growth on described conditions (Harmsen et al., 1995). PCR products different substrates was tested using a bicarbonate-buffered were purified and concentrated using a QIA Quick kit anaerobic medium as described previously by Stams et al. (Qiagen). Approximately 1 µg purified PCR product was (1993), supplemented with 0n02% yeast extract. The in- used for sequence analysis using the Sequenase T7 oculum size of strain ZT was 1%. Substrates were added sequencing kit (Amersham) according to the manufacturer’s from sterile stock solutions up to a concentration of 10 mM instructions. Infrared Dye 41 (MWG-Biotech) labelled unless stated otherwise. For syntrophic growth tests, 1% of primers 515r, 338f (Lane, 1991) and 968f (Nu$ bel et al., 1996) strain ZT was inoculated in hydrogen pregrown cultures of were used