J. Gen. App!. Microbiol., 41, 99-117 (1995) BRACHYMONAS DENITRIFICANS GEN. NOV., SP. NOV., AN AEROBIC CHEMOORGANOTROPHIC BACTERIUM WHICH CONTAINS RHODOQUINONES, AND EVOLUTIONARY RELATIONSHIPS OF RHODOQUINONE PRODUCERS TO BACTERIAL SPECIES WITH VARIOUS QUINONE CLASSES AKIRA HIRAISHI,* YONG KOOK SHIN,' AND JUNTA SUGIYAMA' Laboratory of Environmental Biotechnology, Konishi Co., Ltd., Sumida-ku, Tokyo 130, Japan 'Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan (Received October 18, 1994; Accepted January 13, 1995) The new strains of aerobic chemoorganotrophic rhodoquinone-containing bacteria previously isolated from activated sludge were studied from taxonomic and phylogenetic viewpoints. These strains were Gram- negative, nonmotile coccobacilli, had a strictly respiratory type of metab- olism with oxygen or nitrate as the terminal acceptor, produced catalase and oxidase, and contained both ubiquinone-8 and rhodoquinone-8 as major quinones. DNA-DNA reassociation studies revealed that the new strains were highly related to each other at hybridization levels of more than 74%, suggesting the genetic coherency of the isolates as a single species. The 16S rRNA gene from one of the isolates, strain AS-P1, was amplified in vitro and sequenced directly. Sequence comparisons and a distance matrix tree analysis revealed that strain AS-P 1 was most closely related to Comamonas testosteroni, a representative of the beta subclass of the Proteobacteria, but the level of sequence similarity between the two appeared to be low enough to warrant different generic allocations. The strains were differentiated from related organisms by a number of pheno- typic and chemotaxonomic properties. Thus, we conclude that the isolates should be placed in a new genus and species of the beta subclass of the Proteobacteria, for which we propose the name Brachymonas denitrificans. Evolutionary relationships of rhodoquinone producers to bacterial species with various quinone classes were discussed on the basis of 16S rRNA sequence information. * Present address and address reprints request to: Dr . Akira Hiraishi, Central Research Laboratories of Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki 210, Japan. 99 100 HIRAISHI, SHIN, and SUGIYAMA VOL. 41 Isoprenoid quinones, represented by ubiquinones and menaquinones, are essen- tial components of the respiratory or photosynthetic electron transport systems of bacterial plasma membranes, and their chemotaxonomic significance as well as their physiological importance has been well recognized (6). Rhodoquinones are the purple-colored derivatives of ubiquinones in which one of the methoxyl group in the quinone ring is replaced by an amino group. The purple quinones were first discovered in the phototrophic bacterium Rhodospirillum rubrum (14), and later proved to occur as major quinone components together with ubiquinones in some other species of the purple nonsulfur bacteria (22) . Interestingly, rhodoquinones are also present in mitochondria of some eukaryotes, such as Astasia longa (3), Euglena gracilis (41), and anaerobic helminths (1, 45). In the anaerobic euka- ryotes, rhodoquinones function as mediators of anaerobic electron transport with fumarate as the terminal acceptor (12, 51, 52). Relationships between rhodo- quinones and dissimilatory fumarate reduction have also been suggested in the phototrophic bacteria (17,43). While the taxonomic and biological significance of rhodoquinones has been studied mainly for species of the purple nonsulfur bacteria among prokaryotes, there has been little information on their occurrence in chemotrophic bacteria. Thus far, only Zoogloea ramigera has been reported to produce rhodoquinones among known species of chemotrophic bacteria (26). In a previous study, several strains of rhodoquinone-containing aerobic chemoorganotrophic bacteria were newly isolated from activated sludge (24). These isolates were Gram-negative, asporogenous, nonmotile coccobacilli or short rods, produced catalase and oxidase, had the ability to grow by nitrate respiration, and contained both ubiquinone-8 (Q-8) and rhodoquinone-8 (RQ-8) as major quinones. They seemed to make up a single taxon at the species or generic level because of their homogeneity in basic phenotypic characteristics as well as quinone composition, but could not be allocated to any known taxa of aerobic chemoorganotrophs. The present study was undertaken to characterize the new rhodoquinone- producing isolates more thoroughly from taxonomic and phylogenetic viewpoints. In this paper, we propose to create a new genus and species for the isolates with the name Brachymonas denitrificans, and we refer to them below by the proposed name. Phylogenetic affiliations of rhodoquinone-producing prokaryotes, including B. denitrificans, and evolution of bacteria with various quinone types are discussed on the basis of 16S rRNA sequence information. MATERIALS AND METHODS Bacterial strains and cultivation. Five strains of Brachymonas denitrificans, AS-P1T (T, type strain), AS152, AS163, AS164, and AS353 (24), were studied. Strains AS-P 1T and AS 152 were isolated from soybean curd waste sludge, strains AS 163 and AS 164 were from a laboratory-scale activated sludge unit acclimated with synthetic sewage, and strain AS353 were from municipal sewage sludge. The 1995 Brachymonas gen. nov., sp. nov. 101 following bacterial strains were used for comparison: Acinetobacter calcoaceticus JAM 12087T; Alcaligenes faecalis JAM 12369T; Alcaligenes denitrificans IAM 12370T; Alcaligenes eutrophus TAM 12368T; Comamonas terrigena IAM 12421T; Comamonas testosteroni IAM 12419T;Paracoccus denitrificans JAM 12479T;Rhodo- ferax fermentans FR2T; Rubrivivax gelatinosus ATCC 17011T; Variovorax para- doxus IAM 12373T; and Zoogloea ramigera IAM 12136T. The strains with IAM and ATCC numbers were obtained, respectively, from the Institute of Molecular and Cellular Biosciences, the University of Tokyo (Tokyo, Japan) and from the American Type Culture Collection (Rockville, MD, U.S.A.). R, fermentans FR2T is a strain of own collection (A.H.) (23). Cells of chemotrophic strains were grown aerobically at 30°C either in a complex medium designated PBY (25) or in a chemically defined medium designated LYS (26). Phototrophic bacterial strains were grown anaerobically at 30°C in MYCA medium (23) in light. Enzyme assays. Cells were harvested from cultures at the late exponential phase of growth, washed with 50 mM phosphate buffer (pH 7.0), and resuspended in this buffer. Cell-free extracts were prepared by disrupting cells for 2 min with an Ohtake ultrasonicator (20 kHz, output power, 150 W) followed by centrifugation at 20,000 X g, and was immediately used for enzyme assays. Succinate dehydrogenase activity was measured spectrophotometrically by the method of Arrigoni and Singer (2) modified by King and Drews (35). Fumarate reductase activity with reduced methyl viologen (MVH) or reduced FMN (FMNH2) were measured spectrophotometrically by the method of Jones and Garland (32) with small modifications (17). One unit of enzyme activity was defined as the amount of enzyme that catalyzed l ,amol substrate per minute at 30°C. Protein was measured colorimetrically by the method of Lowry et al. (36) using crystalline bovine serum albumin as the standard. Phenotypic characterization. Unless otherwise indicated, all test media were incubated at 30°C. Cell morphology was studied for cells grown in PBY medium under a phase-contrast microscope and a scanning electron microscope. Tests for anaerobic growth with different terminal oxidants were performed in 20-ml screw- capped test tubes which were completely filled with PBY medium supplemented with either 20 mM potassium nitrate, 20 mM trimethylamine N-oxide (TMAO), 20 mM dimethylsulfoxide (DMSO), 20 mM fumarate, or 20 mM bicarbonate (plus 10 mM pyruvate or fructose). All these supplements were added to the medium through sterile membrane filters (pore size, 0.2 ,um). Carbon source utilization tests were done in 30-ml screw-capped test tubes containing 10 ml of mineral base RM2 (pH 7.0) (26) supplemented with a neutralized, filter-sterilized organic substrate. The tubes were incubated aerobically on a reciprocal shaker, and the final reading was done after 10 days' incubation. Growth was monitored turbidi- metrically with an ANA-75 spectrophotometer (Tokyo Photoelectric Co., Tokyo, Japan). All other phenotypic tests were performed as described elsewhere (8). Analyses of guinones and fatty acids. Cells were harvested from cultures at the early stationary phase of growth, washed with 1 % saline, and then freeze-dried. 102 HIRAISHI, SHIN, and SUGIYAMA VOL. 41 Quinones were extracted with an organic solvent mixture, purified by thin-layer chromatography, and measured spectrophotometrically (22) . Fatty acids were extracted as their methyl ester derivatives and analyzed by gas-liquid chromatogra- phy as previously reported (26, 29). DNA base composition and DNA-DNA hybridization. Cells growing at the mid-exponential phase of growth were harvested, washed with EDTA-saline (0.15 M EDTA + 0.15 M NaCI, pH 8.5), resuspended in this saline, and stored at - 20°C until use. Chromosomal DNA was extracted and purified by the method of Marmur (37). DNA base composition was determined by high-performance liquid chromatography (HPLC) of nuclease P 1 hydrolysates of DNA as described by Katayama-Fujimura et al. (33) with some modifications (23). DNA-DNA reasso- ciation studies were performed by the