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J. Gen. Appl. Microbiol., 56, 193‒203 (2010) Full Paper Rhizomicrobium palustre gen. nov., sp. nov., a facultatively anaerobic, fermentative stalked bacterium in the class Alphaproteobacteria isolated from rice plant roots Atsuko Ueki,1,* Yumiko Kodama,2 Nobuo Kaku,1 Takuya Shiromura,1 Atsuya Satoh,1 Kazuya Watanabe,2 and Katsuji Ueki1 1 Faculty of Agriculture, Yamagata University, Tsuruoka, Yamagata 997‒8555, Japan 2 Exploratory Research for Advanced Technology (ERATO), JST, University of Tokyo, Bunkyo-ku, Tokyo 113‒8656, Japan (Received September 24, 2009; Accepted December 21, 2009) Facultatively anaerobic bacterial strains (A48T, RR25 and RR54) were isolated from roots of liv- ing rice plants in an irrigated rice-fi eld in Japan. The three strains had identical 16S rRNA gene sequences and showed almost the same phenotypic properties examined. Cells of the strains were Gram-negative, non-spore-forming rods. Reproduction of cells was by binary fi ssion as well as by budding. Cells occurred singly or in pairs arranged angularly. Some cells, including dividing cells, were motile with a single polar fl agellum. Cells developed a polar prostheca (stalk) with a holdfast-like structure and the cell with the stalk budded a daughter cell. The strains were chemoorganotrophs and utilized various sugars as growth substrates. The strains fermenta- tively produced acetate and lactate as well as small amounts of ethanol and H2 from the sub- strates. Growth temperature and pH ranges for growth were 15‒40°C and pH 5.5‒7.3 with opti- mum growth at 30‒35°C and pH 6.8. NaCl concentration range for growth was 0‒1.0% (wt/vol) with an optimum at 0% (wt/vol). Catalase and oxidase activities were not detected. The strains reduced Fe(III) to Fe(II) in the presence of glucose, while they did not reduce nitrate, fumarate, malate or sulfate. The major cellular fatty acids of the strains were C18:1ω7, anteiso-C15:0, iso- C15:0, C16:0 and C18:0. Ubiquinone Q-10 was the major respiratory quinone and the genomic DNA G + C contents were 53.4‒55.4 mol%. Phylogenetic analysis based on 16S rRNA gene sequenc- es placed the strains in the class Alphaproteobacteria and the strains formed a novel deep branch in the phylogenetic trees constructed. Based on the differences in 16S rRNA gene se- quences and phenotypic properties of the novel strains from those of their relatives, we pro- posed that the strains be assigned in the novel genus and species as Rhizomicrobium palustre gen. nov., sp. nov. The type strain of the novel species is strain A48T (= JCM 14971T = DSM 19867T). Key Words—Alphaproteobacteria; anaerobic Gram-negative rods; Fe(III)-reduction; Rhizomicrobi- um; stalked bacterium Introduction * Address reprint requests to: Dr. Atsuko Ueki, Faculty of Ag- In Japan, rice is widely cultivated as the principal riculture, Yamagata University, Wakaba-machi 1‒23, Tsuruoka, Yamagata 997‒8555, Japan. food using irrigated fi elds, which develop a highly re- Tel: 0235‒28‒2866 Fax: 0235‒28‒2846 duced condition in the soil during the fl ooding period E-mail: [email protected] (Takai, 1970; Wassmann et al., 2000). The submerged, 194 UEKI et al. Vol. 56 anoxic rice-fi eld soil harbors anaerobic microbial com- tion solution using a Waring blender (10,000 rpm, munities composed of diverse species of microbes 10 min) under N2. The homogenized root samples including fermentative and anaerobically-respiratory were diluted by one-tenth serial dilution under O2-free (nitrate-, Fe(III)- or sulfate-reducing) bacteria as well as N2 gas fl ow and inoculated to the medium described methanogenic archaea (Boone, 2000). In anoxic rice- below by the anaerobic roll tube method (Holdeman et fi eld soil, plant residue such as rice straw ploughed al., 1977; Hungate, 1966). Colonies formed on the roll into the soil provides major amounts of growth sub- tube agar after incubation for about 1 month were strates for microbes and dense microbial populations picked and the strains were obtained after purifi cation usually develop on it (Kaku et al., 2000). Roots of living using the anaerobic roll tube method. The three strains rice plants also supply substrates for microbes in the used were derived from the roll tubes inoculated with rhizosphere by secreting various compounds such as 10-5 diluted samples of the homogenized root sam- saccharides, amino acids and organic acids or by ples. peeling off the root epidermis and cortex. Substrates Media and cultivation method. The strains were derived from these sources stimulate the microbial ac- isolated by using peptone/yeast extract (PY) medium tivity in the rice-fi eld soil and thereby increase produc- or 1/10 PY medium (the concentrations of both pep- tion and emission of methane from the fi elds as one of tone and yeast extract were decreased to one tenth of the greenhouse gases (Boone, 2000; Kaku et al., 2000; those in PY medium) as basal medium with oxygen- Khalil, 2000; Seiler et al., 1984; Takai, 1970; Wassmann free, 95% N2/ 5% CO2 mixed gas as the headspace as et al., 2000). described by Satoh et al. (2002). PY or 1/10 PY medi- We have isolated various fermentative anaerobes um supplemented with (L-1) 0.25 g each of glucose, from different samples collected from an irrigated rice- cellobiose, maltose and soluble starch as well as 15 g fi eld in Japan and already described some novel spe- agar (Difco) were designated PY4S or 1/10 PY4S agar cies derived from plant residue as well as living rice and used for isolation. The strains were anaerobically roots (Akasaka et al., 2003a, b; Satoh et al., 2002; Ueki maintained on agar slants of PY4S medium. Cells for et al., 2006, 2007). In this study, we have characterized physiological and chemotaxonomic examinations were three strains isolated from living rice roots, which were usually cultivated in PY medium supplemented with assigned to the class Alphaproteobacteria and phylo- 10 g L-1 glucose (PYG). The composition of the B-vita- genetically distant from any recognized species. min mixture was as described previously (Akasaka, 2003b). The strains were cultivated at 30°C under the Materials and Methods anaerobic condition unless otherwise stated. Growth in liquid medium was monitored by changes in OD660. Isolation of strains. Strains A48T (= JCM 14971T = Characterization of the strains. Growth of the DSM 19867T), RR25 (= JCM 14972 = DSM 19866) strains under aerobic conditions was examined by and RR54 (= JCM 14973 = DSM 19865) were isolated plate culture on nutrient agar (Nissui Pharmacy) as from roots of living rice plants (Oryza sativa cv. Haenu- well as R2A agar (Satoh et al., 2002). PY4S and 1/10 T ki, type japonica) in August of 1993 (strain A48 ) and PY4S agar modifi ed to exclude Na2CO3, L-cysteine· 2000 (strains RR25 and RR54) collected from the same HCl·H2O and sodium resazurin were also used. Cell irrigated rice fi eld soil in the Shonai Branch of the morphology was assessed by observation of cells by Yamagata Agricultural Research Center (Tsuruoka, phase-contrast microscopy in addition to observation Yamagata, Japan) during the intermittent irrigation pe- of Gram-stained cells by light-microscopy. Cell mor- riod (Satoh et al., 2002). Cultivation practices for rice phology was also observed using transmission elec- plants and other conditions of the fi elds were described tron microscopy to confi rm the presence of fl agella previously (Ueki et al., 2000). The rice plants were tak- and stalks (Kodama and Watanabe, 2004). Growth of en by digging out the roots together with the surround- cells exposed to 80°C for 10 min was examined to ing soil layer using a trowel, and then soil adhering to check the presence of heat-tolerant cells. Oxidase and the roots was removed by washing several times with catalase activities were determined according to the a sterilized, anaerobic dilution solution (Akasaka et al., methods described by Akasaka et al. (2003b). Utiliza- 2003a; Satoh et al., 2002). The root samples were cut tion of carbon sources was tested in PY liquid medium to about 1-cm pieces and homogenized with the dilu- with each substrate added at 10 g L-1 (for sugars and 2010 Rhizomicrobium palustre gen. nov., sp. nov. 195 sugar alcohols) or 30 mM (organic acids, amino acids cluded before sequence assembly. and alcohols). Fermentation products (fatty acids, al- cohols and H2) were analyzed by gas-chromatography Results (GC) as described previously (Akasaka et al., 2003a; Satoh et al., 2002; Ueki et al., 1986). Nitrate-reducing Various phenotypic characteristics were determined activity was determined in PY liquid medium supple- for all three strains (A48T, RR25 and RR54) cultivated -1 -1 mented with 2 g L NaNO3 using glucose (2 g L ), under the anaerobic conditions unless otherwise stat- acetate, lactate, methylamine, methanol or ethanol ed, and they showed similar properties for all charac- (20 mM each) as a possible electron donor. The Fe(III)- teristics examined. Out of the characteristics of the reducing ability was examined with ferric ammonium strains shown below, detailed descriptions such as citrate (20 mM each) as an Fe(III) source using glucose amounts of fermentation products and the composi- (2 g L-1), acetate, pyruvate, lactate, methanol, ethanol tion of CFAs will be mainly based on the data from or L-serine (20 mM each) as a possible electron donor. strain A48T as a representative strain. The concentrations of Fe(II) formed in the medium were determined with the ferrozine method (Stookey, Cell morphology and colony 1970). Malate-, fumarate- and sulfate-reducing activi- The three strains grew under anaerobic conditions ties were determined according to the methods de- by using PY medium as basal medium.

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