Rhizomicrobium Palustre Gen. Nov., Sp. Nov., a Facultatively Anaerobic, Fermentative Stalked Bacterium in the Class Alphaproteobacteria Isolated from Rice Plant Roots

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 living 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 fermentatively produced acetate and lactate as well as small amounts of ethanol and H2 from the substrates. Growth temperature and pH ranges for growth were 15‒40°C and pH 5.5‒7.3 with optimum 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 sequences 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 sequences and phenotypic properties of the novel strains from those of their relatives, we proposed 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, alcohols 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. Colonies on scribed by Satoh et al. (2002). Other characterizations PY4S agar were translucent and thin with a smooth were performed according to the methods described surface. Reduction of concentrations of peptone and by Holdeman et al. (1977) and Ueki et al. (2006). yeast extract in PYG medium (1/10 PYG) did not affect Chemotaxonomical analyses. Whole-cell fatty ac- the anaerobic growth of the strains. Cells of the strains ids (CFAs) were converted to methyl esters (Miller, were Gram-negative rods, 0.3‒0.5 μm in width and 1982) and analyzed by GC (Hp6890, Hewlett-Packard 1.0‒1.5 μm in length. Cells after 2‒4 days of cultivation or G-3000, Hitachi) equipped with a HP Ultra 2 col- (in PY4S slant cultures or PYG liquid medium) were umn. CFAs were identifi ed by equivalent chain-length straight rods with tapered ends as observed by phase- (ECL) (Miyagawa et al., 1979) according to the proto- contrast microscopy, and showed snapping-like divi- col of TechnoSuruga Co., Ltd. (Shimidu, Japan) based sions to produce an angular arrangement of two cells on the MIDI microbial identifi cation system (Microbial (Fig. 1A). Only a small number of cells, even in rela- ID) of Moore (Moore et al., 1994). Isoprenoid quinone tively younger cultures, were motile singly or in pairs was extracted as described by Komagata and Suzuki under the phase-contrast microscope, and the num- (1987) and analyzed by using a mass spectrometer ber of motile cells decreased further in older cultures. (JMS-SX102A; JEOL). G + C content of genomic DNA Transmission electron microscopy confi rmed that was measured by HPLC (Hitachi L-7400) with a cells, as a single cell or one of the pair, had a polar μBondapak C18 column (3.9 × 300 mm; Waters) (Su- fl agellum (up to 4 μm long) (Fig. 2A, B). Cells in pairs zuki et al., 2009). were connected by a constriction, showing sharply- Phylogenetic analysis. Almost the full length of the pointed ends after division (Fig. 2C). It was confi rmed 16S rRNA gene was PCR amplifi ed using the primer by electron microscopy that about 30% of cells in the set of 8f and 1546r. The PCR-amplifi ed 16S rRNA gene 4-day culture had prosthecae on one pole of the cells. was sequenced by using an ABI Prism BigDye Termi- The length of the prosthecae ranged from 0.2 to about nator Cycle Sequencing ready reaction kit and ABI 1.5 μm with a constant diameter of 0.1‒0.15 μm (Fig. Prism 3730 automatic DNA sequencer (Applied Bio- 2C‒H). Some prosthecae swelled at the ends to de- systems). Multiple alignments of the sequences with velop holdfast-like structures (Fig. 2F). The sizes of references in GenBank were performed with the BLAST cells in pairs on the prosthecae were usually unequal, program (Altschul et al., 1997) as well as the FASTA indicating that the cells without the prosthecae were alignment tool (Pearson and Lipman, 1988). A phylo- budded from the cells having the prostheca (Fig. genetic tree was constructed with the neighbor-joining 2E‒H). The daughter cells became fusiform and were method (Saitou and Nei, 1987) by using the CLUSTAL connected to the mother cells with slender constric- W program (Thompson et al., 1994). All gaps and un- tions before separation (Fig. 2H). Based on the obser- identifi ed base positions in the alignments were ex- vations, the prosthecae were considered to be stalks, 196 UEKI et al. Vol. 56

Fig. 1. Photomicrographs of cells of strain A48T. A‒C, cells observed by phase-contrast microscopy; anaerobically cultivated cells for 4 (A) and 7 (B) days, and aerobically cultivated cells for 7 days (C). D‒F, Gram-stained cells; culti- Fig. 2. Transmission electron microscopy of cells cultivated vated anaerobically for 2 (D), 5 (E) and 7 (F) days. Bar, 5 μm. anaerobically for 4 days, showing a polar fl agellum (A and B), binary fi ssion (B and C), development of a polar stalk (C‒H), but not hyphae. The presence of stalks on the cells budding (E‒H), holdfast (F) and constriction connecting divid- could not be certainly confi rmed by phase-contrast ing cells (C and H). A C, strain A48T; D G, strain RR54; H, strain RR25. Bar, microscopy (Fig. 1A, B). When Gram-stained cells ‒ ‒ 0.5 μm. were observed by light-microscopy (Fig. 1D‒F), however, the stalk-like structures of the cells could be confi rmed for the 5-day culture (Fig. 1E). The number of formation was observed upon the addition of 3% stalked-cells seemed to decrease in the older cultures (vol/vol) H2O2 solution to cells cultivated under aerobic (Fig. 1F). conditions. Oxidase activity was not detected. The Although the strains grew in air weakly either on strains grew fermentatively and utilized arabinose, xy- PYG or nutrient agar, the aerobic growth was not im- lose, fructose, galactose, glucose, rhamnose, cellobi- proved in several other media examined including ose, lactose, maltose, sucrose, soluble starch and xy- 1/10 PY4S agar, R2A and the diluted nutrient agar. lan as growth substrates. The strains did not use Cells cultivated in the aerobic condition had the same ribose, mannose, sorbose, trehalose, raffi nose, cellu- morphological features as described above for the lose, glycogen, erythritol, glycerol, inositol, mannitol, cells cultivated in the anaerobic conditions, although sorbitol, citrate, fumarate, lactate, malate, pyruvate, the aerobically-grown cells seemed to be slightly more succinate, L-alanine, L-aspartate, L-glutamate, glycine, slender than the anaerobically-grown cells (Fig. 1C). L-serine, esculin, methanol, ethanol or propanol. Fer- Cultivation under the light did not affect the color of the mentation products in PYG medium were acetate colonies in either condition. Spores were not observed, (6.2 mmol L-1), lactate (10.4 mmol L-1), ethanol -1 -1 and the strains did not produce heat-tolerant cells. (6.7 mmol L ) and H2 (0.19 mmol L ), and fi nal pH Growth of the strains was not affected by the presence grown in PYG medium was 4.8. The strains produced of a B-vitamin mixture in PYG medium. the same fermentation products from all substrates used, although the amounts of each product were Physiological characteristics and substrate spectra slightly different according to the substrate. The strains Catalase activity was not detected in the cells grown reduced Fe(III) (ferric ammonium citrate) to Fe(II) rath- under anaerobic conditions, while very slight bubble er actively in the presence of glucose. Other possible 2010 Rhizomicrobium palustre gen. nov., sp. nov. 197 electron donors examined (acetate, lactate, pyruvate, 18633T (88.0%) in the Hyphomicrobiaceae; Methylosi- methanol, ethanol and L-serine) were not used for nus trichosporium NCIMB 11131T (88.0%) and Methy- Fe(III)-reduction. Nitrate was not reduced when exam- locystis parvus NCIMB 11129T (87.9%) in the Methylo- ined with glucose, acetate, lactate, methylamine, cystaceae; Afi pia birgiae CIP 106344T (88.2%) in the methanol or ethanol as a possible electron donor. The Bradyrhizobiaceae; Pseudoxanthobacter soli DSM strains did not reduce malate, fumarate or sulfate. The 19599T (88.2%) in the Xanthobacteraceae. The closest strains were negative for the production of urease, hy- relatives of the isolates in the Sphingomonadales were drogen sulfi de and indole. Gelatin was not hydro- Sphingomonas melonis DSM 14444T and Sphingomo- lyzed. nas aquatilis KCTC 2881T (87.3% for both) in the Sphin- gomonadaceae (Arun et al., 2008; Garrity et al., 2005; Growth conditions Okamura et al., 2009). The strains grew at 15‒40°C and pH 5.5‒7.3 with optimum growth at 30‒35°C and pH 6.8. Addition of NaCl Discussion to PYG medium signifi cantly decreased the growth rates of the strains, and the concentration range of The novel three strains were isolated from rice plant NaCl for growth was 0‒1.0% (wt/vol). roots of the same irrigated rice-fi eld at two different times with an interval of 7 years. The submerged rice- Chemotaxonomic characteristics fi eld soil is anoxic and a highly reduced condition usu- The three strains had almost the same profi les of ally develops in it. Although the rice roots may remain cellular fatty acid composition. For strain A48T, the ma- relatively oxic with oxygen transported through the jor CFAs were C18:1ω7 (29.4%), anteiso-C15:0 (16.2%), aerenchyma of plants, it was shown that many anaer- C16:0 (13.2%), iso-C15:0 (11.2%) and C18:0 (9.1%). CFAs obes including methanogens were present on rice such as C14:0 (2.4%), 3-OH C14:0 (1.4%), 3-OH iso-C15:0 roots in submerged soil (Kaku et al., 2000; Satoh et al., (1.9%), 3-OH C16:0 (2.6%), iso-C17:0 (1.3%), 2-OH C17:0 2002). Fe(III)-reduction usually proceeds actively in (1.2%) and C18:1ω9 (1.1%) were detected as minor submerged rice-fi eld soil and Fe(II) accumulated in the components. The predominant respiratory quinone of soil acts as a reducing agent in the soil to maintain the the strains was ubiquinone Q-10 (H0). The G + C con- strictly anaerobic, methanogenic environment (Ueki et tents of genomic DNA were 55.4 mol% (strain A48T) al., 2000). It is known that Fe(III)-reduction also occurs and 53.4 mol% (strain RR54). For strain RR25, the re- on rice plant roots in submerged soil and a layer of spiratory quinone composition and the G + C content Fe(II)-deposition develops on rice roots. The bacterial were not determined. group related to the three strains should live by fermentation using various saccharides supplied from Phylogenetic affi liation roots or by Fe(III)-reduction, and these abilities of the The three strains had identical 16S rRNA gene se- three strains should be important to understand the quences and the strains were assigned to the class bacterial ecology of rice roots. The closest strain Alphaproteobacteria (Garrity et al., 2005). The closest (Mfc52) was derived from biofi lm on the anode of a relative on the database was strain Mfc52, isolated microbial fuel cell operating with rice fi eld soil as a from a biofi lm attached to the anode graphite of a mi- source of microorganisms. The cells of strain Mfc52 crobial fuel cell (Kodama snd Watanabe, 2008) with a also possessed prosthecae-like appendages, which sequence similarity of 97.5%. The next closest relative were considered to play an important role in the gen- was a clone, RB521 (AB240224) with a sequence sim- eration of electricity (Kodama and Watanabe, 2008). ilarity of 96.8%, derived from the rhizosphere biofi lm of The closest clone (RB521) was also derived from the a reed bed reactor. rhizosphere biofi lm of reeds in the laboratory. These The three strains were placed at almost equal dis- facts suggest that bacterial groups related to the three tances from many described species in different fami- novel strains should be common and important mem- lies of the Rhizobiales as follows: Hyphomicrobium bers of microbial communities in biofi lm on solid sur- hollandicum ATCC 27498T (sequence similarity based faces under submerged anoxic conditions. on the FASTA search, 88.2%), Blastochloris sulfoviridis Some characteristics of the novel three strains were DSM 729T (88.0%) and Rhodoplanes serenus DSM compared with those of some related genera in the 198 UEKI et al. Vol. 56

Rhizobiales as well as the genus Sphingomonas in the 2005), Maricaulis (Abraham et al., 1999, 2002), Oce- Sphingomonadales (Table 1). Based on the observa- anicaulis (Strömpl et al., 2003) and Woodsholea (Abra- tions of cells by transmission electron microscopy, we ham et al., 2004). The three strains, however, are also confi rmed that reproduction of our strains is by binary only remotely related to any of the members of these fi ssion as well as by budding from mother cells. Fur- stalked bacteria; similarities of 16S rRNA gene se- thermore, we concluded that the polar prosthecae quences between A48T and Caulobacter vibrioides produced by the cells are stalks, but not hyphae, since DSM 9893T, Maricaulis maris ATCC 15268T, Oceani- the daughter cells budded directly from the mother caulis alexandrii DSM 11625T and Woodsholea mari- cells having the prosthecae. The Hyphomicrobium time LMG 21817T are 85.0, 86.1, 83.3 and 84.4%, re- species, one of the closest relatives of the three strains, spectively. are aerobic, and the cell produces a polar hypha de- Except for anoxic, phototrophic bacteria, most of the veloping a daughter cell on the tip (Garrity et al., 2005). species in the class Alphaproteobacteria are aerobic Blastochloris species in the Hyphomicrobiaceae are and have a strictly respiratory type of metabolism with photoheterotrophic under anoxic conditions in the oxygen as the terminal electron acceptor. Only restrict- light, and the mother cell of Blastochloris viridis also ed species in the genera such as Gemmobacter and forms a daughter cell at the end of a slender prostheca Ketogulonicigenium in the order Rhodobacterales, Zy- (Hiraishi, 1997). Rhodoplanes species are also photo- momonas in the order Sphingomonadales and Ancalo- heterotrophic and multiply by budding and asymmet- microbium in the family Hyphomicrobiaceae are de- ric cell division, and the presence of a division tube scribed to be facultatively anaerobic and fermentative. between the mother and daughter cells has been con- Gemmiger formicilis in the family Hyphomicrobiaceae fi rmed for Rhodoplanes serenus (Okamura et al., seems to be the only strictly anaerobic, fermentative 2009). Thus, the novel strains have the feature of pro- species in the class Alphaproteobacteria. Although the ducing polar prosthecae in common with some rela- novel three strains grew under the aerobic conditions, tives in the Hyphomicrobiaceae; however, the function the growth was much weaker than that under anaero- of the prosthecae of the novel strains seems to be dif- bic conditions and the cells of the strains did not have ferent from those of the related species. Cells of other a distinct catalase activity irrespective of the cultivation relatives in the Rhizobiales and the genus Sphingomo- conditions. Many species in the class Alphaproteobac- nas in the Sphingomonadales do not produce any teria have the ability of nitrate reduction, while our prostheca-like structure. In addition, all these organ- strains did not reduce nitrate. The facultatively anaero- isms are aerobic and other physiological properties bic, fermentative and Fe(III)-reducing properties of the are basically different from those of the three strains. three strains are rather unique in all species in the

The novel strains had C18:1 as the dominant CFA, class Alphaproteobacteria including the phylogeneti- which is usually present in most of the species in the cally and phenotypically related species (Garrity et al., class Alphaproteobacteria as a major CFA, while the 2005). profi les of CFAs containing branched-C15:0 as one of Classifi cation of species belonging to the class Al- major CFAs were distinct in those of the relatives. The phaproteobacteria is fundamentally based on the 16S G + C contents of the strains were signifi cantly lower rRNA gene sequences and the class is currently di- than those of the related organisms (Garrity et al., vided into nine orders (Cho and Giovannoni, 2003; 2005). Garrity et al., 2005; Kurahashi et al., 2008; Kwon et al., The class Alphaproteobacteria includes some spe- 2005), most of which consist of morphologically and cies other than the Hyphomicrobium species that form physiologically heterogeneous species. Phylogenetic polar prosthecae (stalks or hyphae) on the cells. The trees including the three strains and the type species known species producing polar prosthecae are cur- of representative genera in the nine orders in the class rently assigned to different orders, that is, Rhizobiales, Alphaproteobacteria (Garrity et al., 2005; Lee et al., Rhodobacterales and Caulobacterales (Garrity et al., 2005) were constructed with the neighbor-joining 2005) or Rhizobiales and Caulobacterales (Lee et al., method (Saitou and Nei, 1987) (Fig. 3). Although the 2005). The morphological features of the novel three topology of the trees slightly varied depending on the strains with polar stalks on the cells resemble those of species selected, the novel three strains always formed the species in the genera Caulobacter (Garrity et al., a deep branch together with strain Mfc52 and clone 2010 Rhizomicrobium palustre gen. nov., sp. nov. 199 , , 68 18:1 14:0 2-OH 16:0 59 ‒ Q-10 Sphingomonas C C C Sphin- gomonadaceae , , 18:1 19:0 cyclo 16:0 Alphaproteobacteria . C Q-10 Pseudoxantho- bacter C C Xanthobacte- raceae pia 69 68.4 , , 18:1 19:1 17:0 cyclo Aerobic Aerobic Aerobic 61.5 ‒ branched- C C --- Q-10 C Bradyrhizobi- aceae Pseudoxanthobacte r are from Arun et al. (2008). 67 18:1 Aerobic, obligately metha- notrophic C Q-8 Methylocystaceae 67 61 ‒ 18:1 Aerobic, obligately metha- notrophic 62 ‒ ----- ++ C , RR25 and RR54) from other related genera in the class T 16:0 , C 70 18:1 Phototrophic (anoxic), chemotrophic (oxic), nitrate reduction Buddingbudding Binary, Binary division Binary division Binary division Binary division Q-10, RQ-10 Q-8 C 71 67 ‒ 18:1 Blastochloris Rhodoplanes Methylosinus Methylocystis Afi Phototrophic (anoxic), chemotrophic (micro-oxic) Polar growth, Polar budding -- C Q-8/9/10, MK-7/8/9 Hyphomicrobiaceae 65 66 ‒ 18:1 Aerobic, nitrate reduction Hypha Hypha (Division tube) Polar growth, Polar budding 59 ‒ + Hyphomicro- bium C Q-9 , 18:0 , anteiso- , C 15:0 , RR25, T 55 18:1 15:0, 16:0 anaerobic, fermentative, reduction Fe(III) binary division 53 ‒ C C - RR54 iso-C A48 Table 1. Some characteristics that differentiate the three strains (A48 Table Family compounds 1 Strain or genus Utilization of C ProsthecaGrowth Stalk Facultatively Major CFAs C Cell division Budding, G+C (%) of genome DNA Quinone Q-10 +, Positive; - , negative. +, Positive; Data except for the genus Pseudoxanthobacter are taken from Garrity et al. (2005), and data for the genus 200 UEKI et al. Vol. 56

Fig. 3. Neighbor-joining tree based on 16S rRNA gene sequences including the three novel strains (A48T, RR25 and RR54) and the type species of representative genera in the nine orders within the class Alphaproteobacteria (Cho and Giovannoni, 2003; Garrity et al., 2005; Kurahashi et al., 2008; Kwon et al., 2005; Lee et al., 2005). Bootstrap values above 500 based on analysis of 1,000 replicates are shown at branch nodes. Es- cherichia coli ATCC 11775T was used as the outgroup. Bar, estimated difference of 2% in nucleotide sequence positions.

RB521 diverging from a major branch including the analysis and also suggested that the order Caulobacte- families in the order Sphingomonadales. rales should include the family Parvularculaceae with Several signifi cant phylogenic analyses of the class the abandonment of the order Parvularculales, which Alphaproteobacteria have been carried out recently was designated mainly on the 16S rRNA sequence based on different standpoints and various data have (Cho and Giovannonl, 2003). Gupta and Mok (2007) shown that further reconstruction of the higher taxo- have also presented just the same conclusion based nomic groups in the class must be established. Lee et on the comprehensive phylogenic analyses of genome al. (2005) proposed that the order Rhodobacterales sequences for many species in the class Alphapro- (Garrity et al., 2005) should be eliminated and the fam- teobacteria. These reports especially suggested that ily ‘Rhodobacteraceae’ be included in the order Cau- the species producing prostheca in the class Al- lobacterales, based on the comprehensive analysis of phaproteobacteria should be reclassifi ed using more 16S rRNA gene sequences in the class. Furthermore, diverse sequence data than the 16S rRNA gene. Badger et al. (2005) indicated that although 16S rRNA Based on the differences in 16S rRNA gene se- gene sequence analysis supported the affi liation of quences and phenotypic properties of the three strains Hyphomonas neptunium with the order Rhodobacter- from those of phylogenetically and phenotypically re- ales, phylogenies based on the sequences of the 23S lated species, we considered that the three strains rRNA gene and three proteins supported the grouping were not validly assigned to any of the families or or- of the genus Hyphomonas as a member of the Cau- ders currently recognized in the class Alphaproteobac- lobacterales. Badger et al. (2006) further indicated, teria. Taking into consideration the above-mentioned based on the genome sequence of H. neptunium, that various reports for the phylogeny of the species in the the species shared more genes with Caulobacter cres- class of Alphaproteobacteria, however, we propose centus than it did with the closer relative according to here that the strains be assigned in a novel genus and the 16S rRNA phylogeny. Williams et al. (2007) reached species as Rhizomicrobium palustre gen. nov., sp. nov. the same conclusion according to the multiprotein tree without defi nition of the higher taxonomic affi liation in 2010 Rhizomicrobium palustre gen. nov., sp. nov. 201 the class Alphaproteobacteria. The type strain of the to Fe(II) in the presence of glucose. Does not reduce novel species is A48T (= JCM 14971 T = DSM 19867T) nitrate, fumarate, malate or sulfate. Does not produce isolated from rice plant roots in irrigated rice fi eld soil indole, hydrogen sulfi de or urease. The major cellular in Japan. Strain RR25 (= JCM 14972 = DSM 19866) fatty acids are C18:1ω7, anteiso-C15:0, iso-C15:0, C16:0 and RR54 (= JCM 14973 = DSM 19865) as reference and C18:0. The genomic DNA G + C contents are strains were also derived from rice roots of living rice 53.4‒55.4 mol%. The type strain of the novel species is plants obtained from the same fi eld in a different year. A48T (= JCM 14971T = DSM 19867T) isolated from The GenBank/EMBL/DDBJ accession numbers for the rice plant roots in fl ooded rice fi eld soil. Strain RR25 16S rRNA gene sequences of strains A48T, RR25 and (= JCM 14972 = DSM 19866) and strain RR54 (= JCM RR54 are AB081581, AB174809 and AB174822, re- 14973 = DSM 19865) as reference strains are derived spectively. from roots of living rice plants of the same fi eld collected in different years. Description of Rhizomicrobium gen. nov. Rhizomicrobium (Rhi.zo.mi.cro’bi.um. Gr. n. rhiza a Acknowledgments root; N.L. neut. n. microbium a microbe; N.L. neut. n. This work was partly supported by a Grant-in-Aid from the Rhizomicrobium microbe living in a root or rhizo- Institute for Fermentation, Osaka. We are grateful to M. Wa- sphere). tanabe, K. Murasawa and Y. Ohtaki for their technical assistance Cells are Gram-negative, non-spore-forming, motile in characterization of the strains. rods. Cells divide by binary fi ssion as well as by budding and have polar stalks. The major respiratory qui- References none is ubiquinone Q-10. The type species is Rhizomi- crobium palustre. Abraham, W.-R., Strömpl, C., Bennasar, A., Vancanneyt, M., Snauwaert, C., Swings, J., Smit, J., and Moore, E. R. B. Description of Rhizomicrobium palustre sp. nov. (2002) Phylogeny of Maricaulis Abraham et al. 1999 and proposal of Maricaulis virginensis sp. nov., M. parjimensis Rhizomicrobium palustre (pa.lu.st’re. L. neut. adj. sp. nov., M. washingtonensis sp. nov. and M. salignorans palustre living in swamps). sp. nov. Int. J. Syst. Evol. Microbiol., 52, 2191‒2201. 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