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Aminobacterium Mobile Sp. Nov., a New Anaerobic Amino-Acid-Degrading Bacterium

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Aminobacterium Mobile Sp. Nov., a New Anaerobic Amino-Acid-Degrading Bacterium International Journal of Systematic and Evolutionant Microbioloav (2000). 50,259-264 Printed in Great Britain Aminobacterium mobile sp. nov., a new anaerobic amino-acid-degrading bacteriym ier,l P"fL""""J.-L.lGarcial P and B. K. C. Pate Author for correspondence: B. K. C. Patel. Tel: $61 417 726671. Fax: $61 7 38757656. e-mail : [email protected] 1 Laboratoire ORSTOM de A novel, curved (0.3 x 4-0-5.0 pm), Gram-negative, non-sporulating, mesophilic Microbiologie des bacterium, designated strain ILE-3T0 =type strain), was isolated from an Anadrobies, Universitd de Provence, CESBIESIL Case anaerobic lagoon in a dairy wastewater treatment plant. Optimal growth 925,163 Avenue de occurred at 37 OC and pH 7.4 on a medium containing serine as an energy Luminy, 13288, Marseille source and yeast extract. The strain was motile by means of one or two lateral Cedex 09, France flagella. It required yeast extract for growth on serine, glycine, threonine and 2 Departamentode Biologia, pyruvate. Poor growth was obtained on cysteine, Casamino acids, biotrypcase, Pontificia Universidad Javeriana, POB 56710, peptone and 2-oxoglutarate. In the presence of Methanobacterium formicicum, Santa Fe de Bogota, strain ILE-3I oxidized alanine, glutamate, leucine, isoleucine, valine and Colombia aspartate to a minor extent. The G+C content of the DNA was 44 mol%. 3 School of Biomolecular Phylogeneticanalysis of the 16s rRNA gene of strain ILE-3Tindicated that it and Biomedical Sciences, was related to Aminobacterium colombiense (95% similarity value). On the Faculty of Science, Griffith University, Nathan, basis of the phenotypic and phylogenetic characteristics, strain ILE-3Tis Brisbane41 11, Australia designated as a new species of the genus Aminobacterium, namely Aminobacterium mobile sp. nov. (= DSM 12262"). Keywords : amino acid utilization, interspecies hydrogen transfer, Methanobacterium formicicum, Aminobacterium mobile Saccharolytic amino-acid-degrading bacteria and the 1998b) and two new genera, Aminobacterium and non-saccharolytic amino-acid-degrading bacteria play Aminomonas (Baena et al., 199Sa, 1999). In this report, a significant role in protein turnover in the rumen we describe the isolation and characterization of a new (Smith & MacFarlane, 1997). The saccharolytic amino member (strain ILE-3T) of the genus Aminobacterium, acid degraders have been rigorously studied in this which we have designated Aminobacterium mobile sp. ecosystem (Hobson & Wallace, 1982; Wallace, 1986). nov. With the isolation of non-saccharolytic amino acid The methods used for the preparation of media have degraders (Chen & Russell, 1988, 1989; Paster et al., been described previously (Baena et al., 199Sa). For 1993; Attwood et al., 1998), much greater attention initiating enrichments, a sludge sample collected from has now been paid to this bacterial community in an anaerobic dairy wastewater treatment lagoon terms of their diversity and their role in the rumen. In (Santa Fe de Bogota, Colombia) was serially diluted contrast, reports on the extent of microbial diversity of 10-fold, inoculated into a basal medium containing such microbes in other environments is scant (Stams & isoleucine (10 mM) and yeast extract (0.2 %) and then Hansen, 1984; Zindel et al., 1988; Baena et al., 1998a, incubated at 37 OC for up to 4 weeks. The same 1999). As part of our investigations, we chose a medium, fortified with agar (2 %), was used to prepare protein-rich, anaerobic dairy wastewater lagoon to roll tubes for the isolation of pure cultures. The study diversity of non-saccharolytic amino-acid-de- methods used for bacterial characterization and co- grading microbes and the role they play in protein culture studies with Methanobacterium formicicum turnover. We had previously reported the isolation (DSM 1535) have been described previously (Baena et and characterization of various obligate amino acid al., 1998a). Growth and product formation were degraders from this environment, including a new analysed after 2 to 3 weeks of incubation at 37 OC. species of sulfate-reducing bacteria (Baena et al., Light- and electron-microscopy methods were used as described by Fardeau et al. (1997a). Growth was I _Il_n_- iI R O111 1 Q 2000 IUMS 259 - I ocumewtaire I B Fonds Documentaire IRD Y /I Crib Rrw 2 lSff;Z Ex : S. Baena and others 1 I measuring the optical density at 58Onm. The fer- 4 mentation end products were determined as described by Fardeau et al. (1993). Amino acid concentrations 26 were measured using HPLC (Moore et al., 1958). &! The G+C content was determined by the DSMZ u I' P (Deutsche Sammlung von Mikroorganismen und !. 2Ja Zellkulturen, Braunschweig, Germany) using the method of Mesbah et al. (1989). For substrate-utilization studies, 0.2 % yeast extract was added to the basal medium. L-Amino acids (serine, I threonine, glycine, cysteine, alanine, glutamate, valine, z isoleucine, proline, methionine, aspartate, leucine, ! phenylalanine, histidine, asparagine, glutamine, ar- , ginine, lysine), organic acids (pyruvate, succinate, malate, fumarate, citrate, 2-oxoglutarate7lactate, acet- I ...................................................... ............. 9 ......................... s....... ...... ......... * ............................ ate, propionate, butyrate) and sugars (glucose, sucrose, Fig. 1. Phase-contrast photomicrograph of cells, from 1: ribose, xylose, cellobiose, melibiose, maltose, galac- exponential growth-phase culture of strain ILE-3T, depicting tose, mannose, arabinose, rhamnose, lactose, sorbose, their curved shape. Bar, 10 fim. mannitol) were each tested at a final concentration of 10 mM. Glycerol and ethanol were added to a final concentration of 5 mM,whereas biotrypcase, peptone, identified as members of the genus Clostridium but Casamino acids, gelatin and casein were each tested at were not studied further. The second type of colony a final concentration of 0.5 %. The electron acceptors was round, whitish and possessed smooth edges. tested were thiosulfate (10 mM), sulfate (10 mM), Microscopic examination revealed the presence of elemental sulfur (2 %), sulfite (2 mM), sodium curved cells in the latter colony; this culture was fumarate (20 mM) and nitrate (10 mM). Amino acid designated ILE-3T and was characterized further. degradation via the Stickland reaction was tested in a Although strain ILE-3T was initially enriched on 'basal medium containing 10 mM alanine as electron I isoleucine and yeast extract, it grew poorly in this donor and 20mM glycine, 20mM serine, 20mM , medium as a pure culture, suggesting that the hydro- -proline or 20 mM arginine as electron acceptor. genotrophic microbial partner (Clostridium sp.) in the DNA extraction and amplification of the 16s rRNA initial enrichments was important for isoleucine degra- gene have been described previously (Andrews & Patel, dation. Accordingly, Co-culture experiments revealed 1996; Baena et al., 1998a). The new sequence data that strain ILE-3T grew on isoleucine only in the generated were aligned, checked for accuracy manually presence of a hydrogen scavenger, i.e. M.formicicum using the alignment editor, ae2 (Maidak et al., 1996), (see below). Therefore, we assume that the strain's and deposited in GenBank. The BLAST program ability to grow in initial enrichment cultures was due to (Altschul et al., 1997) was used against the GenBank the hydrogenotrophic, acetogenic Clostridium strain. database to determine homology with recently released Further studies revealed that strain ILE-3T fermented .closely related sequences. For analysis, the sequences serine (10 mM) in the presence of yeast extract (0.2 %). ' of Anaerobaculum thermoterrenum, Dethiosulfovibrio These 'new culture conditions were uskd for growth- peptidovorans and Aminobacterium colombiense were characterization studies. extracted from GenBank (accession nos U5071 1, Strain ILE-3T cells were Gram-negative, non- U528 17 and AF069287, respectively) and manually sporulating, curved rods (Fig. 1) measuring 4.0- x pm. aligned with the prealigned sequences obtained from 5.0 03 Cells were motile by means of one or two the Ribosomal Database Project (Maidak et al., 1996). lateral flagella. Ultra-thin sections of strain ILE-3T Pairwise evolutionary distances based on 1151 un- revealed a multilayered, complex, Gram-negative cell ambiguous nucleotides were computed using the wall. Strain ILE-3T did not require NaCl for growth, PHYLE package (Felsenstein, 1993) and TREECON (Van grew optimally at a temperature of 37OC (the tem- de Peer & De Wachter, 1993). perature range for growth was 35-42 OC) and a pH 7.4 Positive growth was observed in the dilution tube (the pH range for growth was 6.7-8.3). after 4 weeks incubation at 37 OC in the basal medium Table 1 shows the substrates used by strain ILE-3T and containing isoleucine and yeast extract. 2-Methyl- the end products. Strain ILE-3T required yeast extract butyrate and acetate were detected as the major end for growth. It fermented serine, glycine, threonine and products. Microscopic examination revealed the pres- pyruvate but growth was poor on Casamino acids, ence of two morphotypes, i.e. curved and straight rods. peptone, biotrypcase, cysteine and 2-oxoglutarate. The Subsequently, two distinct types of colonies developed fermentation end products from these substrates in- in the highest serially diluted roll tube after 2 weeks cluded acetate and H,. Propionate and acetate were incubation at 37 OC. One type of colony was rhizoid the end products of 2-oxoglutarate fermentation. and
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