Brachymonas Denitrificans Gen. Nov., Sp. Nov., An

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 metabolism 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 phenotypic 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 eukaryotes, rhodoquinones function as mediators of anaerobic electron transport with fumarate as the terminal acceptor (12, 51, 52). Relationships between rhodoquinones 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 paradoxus 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 chromatography 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 reassociation studies were performed by the quantitative dot blot hybridization method with photobiotin labeling and colorimetric detection as described previously (10, 23). The level of hybridization was calculated by measuring the color intensity of hybridized spots with a Shimadzu model CS-9000 two-dimensional densitometer. 16S rDNA sequencing and phylogenetic analysis. 16S rRNA-specific DNA was amplified by the polymerase chain reaction (PCR) and sequenced directly by a combined method consisting of linear PCR sequencing and automated fluorescence detection as reported previously (20,27). Compiling of sequence data and calculation of pairwise sequence similarity were performed with the GENETYX- MAC program on an Apple Macintosh personal computer. The CLUSTAL V program (16) was used for multiple alignment of sequences, calculation of the nucleotide substitution rate Knu~ (34), and the construction of neighbor-joining distance matrix trees (44). The bootstrap option (11) in this program package was used for statistical analysis of branching patterns on the phylogenetic tree with 1,000 bootstrapped trials. Nucleotide sequence accession numbers. The 16S rDNA sequences determined in the present study have been deposited in the DDBJ, EMBL, GSDB, and NCBI nucleotide sequence databases under the following accession numbers: B. denitrificans, D l4320; Variovorax paradoxus, D30793.

RESULTS AND DISCUSSION

Morphology and cultural properties As already reported in a previous paper (24), the cells of B. denitrificans strains were Gram-negative, nonmotile coccobacilli or short rods measuring 0.6 to 1.0 am in width and 1.2 to 2.O,am in length (Fig. 1). They reproduced by binary fission. The cells occurred singly or in pairs, sometimes in chains. Neither endospores nor resting cells were observed. All strains of B. denitrificans were nutritionally nonexacting and grew well on ordinary nutrient agar containing peptone or mineral agar supplemented with a simple organic compound as carbon and energy sources. The colonies on LYS agar were smooth, convex, opaque, with entire margin, cream, beige, or pale yellow. 1995 Brachymonas gen. nov., sp. nov. 103

Fig. 1. Phase-contrast (A) and scanning electron (B) micrographs showing general morphology of Brachymonas denitrificans AS-P1T• Crala• A lfl ~~m• R him

The diameter of colonies on LYS agar became 2 to 3 mm after 2 days of incubation. In liquid media incubated with shaking, growth occurred as a uniform cell suspension. No flocculent growth occurred at any growth stage.

Growth and metabolism B. denitrificans strains had a strictly respiratory type of metabolism with oxygen as the terminal electron acceptor, as described previously (24). Chemo- organotrophic growth by aerobic respiration was the best growth mode of these strains, and the doubling time with this growth mode was 1.5 to 2.0 h (in PBY or LYS medium). In spite of this property, they produced the low-potential quinones, rhodoquinones, constitutively. This is curious but interesting, because rhodoquinones are known to play a role in fumarate-terminated anaerobic electron transport in anaerobic eukaryotes and in the phototrophic bacteria. In this connection, we investigated whether B. denitrificans strains can grow anaerobically using exo- genous fumarate and some other compounds as terminal oxidants. Table 1 shows the anaerobic growth profiles of B, denitrificans strains with different terminal oxidants added. Among the tested compounds as possible terminal acceptors, only nitrate supported significant growth of all test strains under anaerobic conditions. Anaerobic growth by nitrate respiration was ac- companied with N2 gas production. These results confirm the previous finding (24) concerning the capacity of B. denitrificans for complete denitrification. The doubling time for nitrate-respiring anaerobic growth was 2.5 to 2.8 h (in PBY medium + 20 mM potassium nitrate). Two of the test strains, AS 163 and AS 164, were also capable of growing anaerobically with TMAO. Nevertheless, TMAO- dependent growth was much slower and weaker than the growth by nitrate respiration. Neither fumarate nor bicarbonate supported anaerobic growth of any of the test strains. 104 HIRAISHI, SHIN, and SUGIYAMA VOL. 41

Table 1. Anaerobic growth of Brachymonas denitrificans strains in PBY medium supplemented with different terminal oxidants.

A representative strain of B. denitrificans, AS-P1T, was further examined for quinone contents and succinate dehydrogenase and fumarate reductase activities (Table 2). Strain AS-PlT produced appreciable amounts of both the benzoquinones, ubiquinones and rhodoquinones, regardless of growth conditions. How- ever, cellular rhodoquinone contents varied significantly dependent upon growth conditions, being much lower than in nitrate-respiring cells than in aerobically grown cells. Also, rhodoquinone concentrations were somewhat lower in cells grown in LYS medium compared to those grown in PBY medium. Respiratory enzyme assays showed that fumarate reductase activity with FMNH2 as the electron donor, as well as succinate dehydrogenase activity, was detected in the cell-free extract of strain AS-P 1T. FMNH2-linked fumarate reductase activity was higher in the extract from aerobically grown cells than in that from nitrate-respiring cells. MVH-dependent fumarate reductase was absent. As described above, our attempts to demonstrate fumarate-dependent anaerobic growth of B, denitrificans were unsuccessful, although the cell-free extract of the organism gave positive results for FMNH2-linked fumarate reduction. Among the terminal oxidants tested, only nitrate has been found to support significant anaerobic growth of all strains of this species. However, it is unlikely that nitrate-dependent anaerobic growth of B, denitrificans is associated with rhodoquinones, partly because the rhodoquinone content is markedly reduced in the nitrate-respiring cells and partly because nitrate respiration is a high potential electron transport process in which ubiquinones are usable as electron carriers. It has been reported that some phototrophic bacteria with rhodoquinones are capable of anaerobic-dark growth at the expense of fructose and bicarbonate (18, 48), where the fumarate reduction system possibly works to maintain redox balance (17). Unlike these phototrophic bacteria, B. denitrificans has been shown to be unable to grow anaerobically on fermentable substrates plus bicarbonate. In view of the results presented here, it can be assumed that, in B. denitrificans, a rhodoquinone-mediated electron transport system possibly linked to fumarate reduction functions to dispose of excess reducing power when the availability of oxygen as the terminal oxidant is reduced. Full understanding of the biological roles of rhodoquinones in aerobic chemotrophic bacteria awaits further study. 1995 Brachvmonas refl. nov.. so. nov. los

Table 2. Quinone contents and respiratory activities of Brachymonas denitrificans AS-P1T grown under different conditions.

Other phenotypic characteristics B. denitrificans strains shared many common characteristics in terms of morphology, physiology, and biochemistry as described above and in a previous paper (24). Additional common characteristics they shared were as follows. Growth occurred in a temperature range of 10 to 40°C (optimum, 30-35°C) and in a pH range of 5 to 9 (optimum, pH 7.0-7.5). NaCI was not required for optimal growth, and multiplication occurred up to 3 % NaCI. Negative reactions were found for indole production, Voges-Proskauer reaction, urease, phenylalanine deaminase, carboxylation of arginine, lysine, and ornithine, hydrolysis of starch, alginate, chitin, gelatin, casein, tributyrin, and Tween 80, and acid production from sugars including L-arabinose, D-xylose, L-rhamnose, D-fructose, D-glucose, D- mannose, D-galactose, maltose, cellobiose, lactose, sucrose, melibiose, adonitol, glycerol, mannitol, and sorbitol. Tests for utilization of organic substrates as carbon and energy sources revealed that B. denitrificans strains exhibited a wide assimilation spectrum of organic compounds other than sugars (Table 3). Good carbon sources were acetate, lactate, pyruvate, fumarate, succinate, and some amino acids. Gas chromatographic analyses of whole-cell fatty acids showed that all test strains of B. denitrificans had saturated and monounsaturated straight-chain fatty acids. The percentage distribution of the nonpolar components detected was quite similar among the test strains; the major components were palmitoleic acid (C16:1) (33-43%) and palmitic acid (C16:0) (21-28%). Significant amounts of C 18:1 (10- 17%) were also found. 3-Hydroxy decanoic acid (C10:0)was present as the major 3-OH component in all strains.

Genomic DNA relatedness DNA-DNA relatedness between B. denitrificans and related bacteria is shown in Table 4. The DNAs of all test strains of B. denitrificans had high reassociation levels of more than 74% against labeled DNA from strain AS-P 1Tor strain AS 163, suggesting the genetic coherency of these strains as a single species. The DNA of 106 HIRAISHI, SHIN, and SUGIYAMA VOL. 41

Table 3. Utilization of organic compounds as carbon and energy sources by Brachymonas denitrificans strains. 1995 Brachymonasgen. nov.,sp. nov. 107

strain AS-P1T exhibited much lower levels of hybridization than any test strain of representative species of the Proteobacteria (50). Among the species used for comparison, C. testosteroni, a representative of the beta subclass, showed the highest affinity (28% homology) to B. denitrificans. The morphologically similar species P. denitrificans and A. calcoaceticus, which belong to the alpha and gamma subclasses, respectively, showed most genomic dissimilarities (<5% homology) to B. denitrificans. Previous work has shown that B. denitrificans strains have DNA base ratios of 63.6 to 65.1 mol% (G + C) (24). In this study, we confirmed that the G + C contents of the test strains of this species fell within this range.

16S rDNA sequence comparisons and phylogenetic analysis The 16S rRNA genes from B. denitrificans and a reference organism, V. paradoxus, were amplified by PCR and sequenced directly by cycle sequencing followed by automated fluorescence detection. The determined sequences were 1,452 to 1,457 residues, corresponding to a continuous stretch from positions 28 to 1,491 of the E. coli rRNA (4). The B. denitrificans sequence was compared with a data set consisting of 15 reference sequences derived from the databases and the sequence of V. paradoxus determined in this study. Since the results of quinone profiling and DNA-DNA pairing suggested B, denitrificans to be a member of the

Table 4. Genomic DNA relatedness among strains of Brachymonas denitrificans and related taxa with different quinone types. 108 HIRAISHI, SHIN, and SUGIYAMA VOL. 41 1995 Brachymonas gen. nov., sp. nov. 109 beta subclass of the Proteobacteria as described above, the reference sequences were selected among those of species belonging to this phylogenetic group. Table 5 shows the level of overall percentage similarity for each pair of sequences and the levels of evolutionary distance (KnU~)for the 1,324 positions of the entire sequence set which could be aligned. On the basis of the evolutionary distance values obtained, a neighbor joining phylogenetic tree was reconstructed (Fig. 2). The results shown in Table 5 and Fig. 2 demonstrate that B, denitrificans is most closely related to C. testosterone among the species compared. The tree shows that B. denitrificans, C. testosterone, R, fermentans and V. paradoxus fall into a mono- phyletic cluster (99.9% support of bootstrapping) which may correspond to the family Comamonadaceae (surrounded by a shaded area).

Taxonomic affiliation of B, denitrificans The phylogenetic analysis on the basis of 16S rRNA and rDNA sequences has shown that B. denitrificans is a member of the beta subdivision of the Proteobacteria, most closely related to C. testosteroni. Comamonas species and related members of chemotrophic bacteria with Q-8 have been included in a phylogenetic group, "acidovorans rRNA complex," on the basis of DNA-rRNA hybridization data (9). This group is now assigned to the family Comamonadaceae (55), which includes the genera Acidovorax, Hydrogenophaga, Xylophilus, and Variovorax in addition to the genus Comamonas. Unfortunately, no information is available on overall 16S Comamonadaceae

Fig. 2. Distance matrix tree showing phylogenetic affiliations of Brachymonas denitrificans AS-P1T and related species within the beta subclass of the Proteobacteria. The sequence of Escherichia coil was used as an outgroup. Bootstrap confidence values are indicated at branching points of interest. 110 HIRAISHI, SHIN, and SUGIYAMA VOL. 41 rRNA sequences of any species of the former three genera, and thus, we can not elucidate detailed phylogenetic relationships of B, denitrificans to known members of the Comamonadaceae. The results for genomic DNA relatedness and pairwise 16S rRNA sequence similarities, however, provide circumstantial evidence for the assessment of the phylogenetic and taxonomic position of B. denitrificans. B, denitrificans exhibited hybridization levels of 17 to 28% to the tested species of Comamonadaceae genera. The levels of 16S rRNA sequence similarity between B. denitrificans and C. testosteroni or V. paradoxus are around 95 and 92%, respectively. Thus, Brachy- monas is considered to have marginal relationships to established genera of the Comamonadaceae. Phenotypic and chemotaxonomic data provide more definitive information to warrant the taxonomic status of Brachymonas as a distinct genus. The characteristic features of this genus distinct from known genera of the Comamonadaceae include coccoid cell morphology, nonmotility, denitrification, and production of both Q-8 and RQ-8. Differential properties of the genus Brachymonas and allied genera are shown in Table 6.

Description Brachymonas gen. nov. Brachymonas (Bra.chy.monas. Gr. adj. brachy short; Gr. n. monas, a unit, monad; M.L. fem. n. Brachymonas, a small short unit). Cells are nonsporeform- ing nonmotile coccobacilli or short rods measuring 0.6 to 1.0,um wide and 1.2 to 2.0,am long. Gram-negative. Aerobic chemoorganotroph having a strictly respiratory type of metabolism with oxygen as the terminal electron acceptor. Anaerobic growth with nitrate as the terminal acceptor is also possible; denitrification positive. Mesophilic, neutrophilic, and nonhalophilic. No growth factors are required. Good growth occurs in ordinary nutrient media containing peptone or in mineral media supplemented with simple organic compounds as carbon and energy sources.

Table 6. Differential characteristics of the genus Brachymonas and related genera. 1995 Brachymonasgen. nov.,sp. nov. 111

Catalase and oxidase positive. Amylase, protease, and lipase are absent. No acid is produced from sugars including glucose. Major fatty acid components are C16:1 and C16:0. 3-OH C10:0is present. Major quinones are Q-8 and RQ-8. Mol% G+ C of DNA is from 63 to 65. Phylogenetic position is in the beta subclass of the Proteobacteria, possibly a member of the family Comamonadaceae. The type species is Brachymonas denitrificans.

Brachymonas denitrificans sp. nov. Brachymonas denitrificans (de.ni.tri'fi.cans. M. L. part. adj. denitrificans deni- trifying). The characteristics are the same as those described for the genus. Additional characteristics are as follows. Colonies on ordinary nutrient agar are smooth, convex, opaque, with entire margin, cream or pale yellow. Growth occurs in a temperature range of 10 to 40°C (optimum, 30-35°C) and in a pH range of 5 to 9 (optimum, pH 7.0-7.5). NaCI is not required for optimal growth, and multiplication occurs up to 3 % NaCI. The following properties are negative: indole production, Voges-Proskauer reaction, urease, phenylalanine deaminase, carboxylation of arginine, lysine, and ornithine, hydrolysis of starch, alginate, chitin, gelatin, casein, tributyrin, and Tween 80, and acid production from sugars including L-arabinose, D-xylose, L-rhamnose, D-fructose, D-glucose, D-mannose, D- galactose, maltose, cellobiose, lactose, sucrose, melibiose, adonitol, glycerol, mannitol, and sorbitol. Good carbon and energy sources are acetate, lactate, pyruvate, fumarate, succinate, alanine, glutamate, leucine, phenylalanine, and proline. Other usable carbon sources are: butyrate, malate, glutarate, benzoate, ethanol, and polypropylene glycol. Not utilized are: formate, caproate, caprylate, peragonate, citrate, gluconate, malonate, tartrate, xylose, arabinose, rhamnose, fructose, glucose, mannose, cellobiose, sucrose, lactose, adonitol, dulcitol, mannitol, sorbitol, methanol, aminobutyrate, glycine, arginine, ornithine, lysine, tryptophan, and histidine. Source: Activated sludge. Type strain: AS-P 1, which has been deposited with the Japan Collection of Microorganisms, RIKEN (Wako, Japan) as Brachymonas denitrificans JCM 9216.

Evolutionary relationships of rhodoquinone producers to bacteria with various quinone classes Species of rhodoquinone-producing organisms have been limited mostly to those of the phototrophic purple nonsulfur bacteria among prokaryotes. A previous study on systematic surveys of rhodoquinones in the phototrophic bacteria has shown that the following six species are rhodoquinone producers: Rhodoferax fermentans [formerly referred to as the "RGL" group (23)], Rhodomicrobium vannielii, Rhodopila globiformis, Rhodopseudomonas acidophila, Rhodospirillum rubrum, and Rhodospirillum photometricum (22). Recently, Rhodopseudomonas rosea, which was proposed to be transferred into the new genus Rhodoplanes, has also been shown to produce rhodoquinones (28). Phylogenetic analyses by 16S 112 HIRAISHI, SHIN, and SUGIYAMA VOL. 41 rRNA cataloging (58) and sequencing (21,28,57) have shown that all these members, except Rhodoferax fermentans, belong to the alpha subclass of the Proteobacteria. On the other hand, the species of chemotrophic rhodoquinone producers so far described are limited to Z. ramigera and B, denitrificans, both of which are proteobacterial members of the beta subclass, as reported here and elsewhere (47). The occurrence of rhodoquinones in those restricted members of phototrophs and chemotrophs is of particular interest in conjunction with the evolution of the rhodoquinone producers and rhodoquinone-associated electron transport systems. To provide insight into this problem, we studied phylogenetic affiliations of rhodoquinone-containing bacteria among members of the domain Bacteria with various quinone classes. Another phylogenetic tree was reconstructed on the basis of Knu~ values obtained for 1,115 positions of the sequence set of all rhodoquinone-producing species and representative bacterial members of different phylogenetic groups (division or class) (57) with various quinone classes (Fig. 3). The topology of the

Fig. 3. Distance matrix tree showing phylogenetic relationships of rhodoquinone producers to bacterial members with various quinone types. The sequence of Thermotoga maritima was used as an outgroup. Thick solid lines show the lineages of members with the naphthoquinones as the sole quinones, single solid lines show those of members with both naphthoquinones and benzoquinones, and dashed lines show those of members with the benzoquinones only. The lines of descent of rhodoquinone producers are also shown by arrows. Information on quinone profiles from the following Refs. 15) for C aurantiacus; 38) for S heparinum; 49) for P. limnophilus; 7) for D. desulfuricans; 5) for C jejuni; 19) for H. chlorum; 47) for Z. ramigera; 46) for E. longus; 22, 28, 30) for the species of phototrophic purple bacteria; and 6) for all other species shown. 1995 Brachymonas gen, nov., sp. nov. 113 tree shows that members of green nonsulfur bacteria, thermophilies, flavobacteria, green sulfur bacteria, planctomycetes, and Gram-positive bacteria, all of which contain menaquinones and/or their analogs as the sole components, branched off deeper than members with ubiquinones, i.e., those belonging to the alpha, beta, and gamma subdivisions of the Proteobacteria. Although Desulfovibrio desulfuricans and Campylobacter jejuni are reported to form the delta and epsilon subdivisions, respectively, of the Proteobacteria, they are located outside the major proteobacterial cluster and are linked to the green sulfur bacteria as the phylogenetic neighbor. Considering the fact that D, desulfuricans and C, jejuni contain menaquinones as the sole quinones (5, 7), however, this branching pattern seems more consistent. The tree topology confirms that rhodoquinone-producing bacteria are distributed among members of the alpha and beta subdivisions of the Proteobac- teria. Since ubiquinones are precursors of rhodoquinones in their biosynthetic process (40, 42), the former quinones are considered evolutionarily older than the latter. Therefore, it is likely that rhodoquinone-containing bacteria appeared after proteobacterial members of each subdivision with ubiquinones branched off from their common ancestor. That is, rhodoquinone producers of each subdivision have evolved independently. Nevertheless, one can not exclude the possibility that genetically different organisms have acquired such a property as rhodoquinone

Fig. 4. Hypothetical illustration for evolution of bacterial quinones with energy- transducing systems. MK, menaquinone; K,, vitamin K,; Q, ubiquinone; PQ, plastoquinone; RQ, rhodoquinone; FRD, fumarate r`eductase; SDH, succinate dehydrogenase. 114 HIRAISHI, SHIN, and SUGIYAMA VOL. 41 production by lateral gene transfer. In this connection, B. denitrificans can be considered one of the key organisms in investigating the evolution of rhodoquinone- involved phenotypes, because this species is physiologically and genetically different from most of the rhodoquinone-producing proteobacteria. On the basis of the phylogenetic analysis of bacteria with various quinone classes as noted above, together with previous reviews of Gest (13) and Jones (31) for the evolution of bacterial energy metabolism, we propose a hypothetical illustration concerning the evolution of bacterial quinones and energy-transducing systems with fumarate reduction (Fig. 4). It is speculated that early accessory oxidant systems used for reoxidation of NADH were relatively simple and soluble without any type of quinones, and functioned only to ensure redox balance, where the use of fumarate as a terminal oxidant was an important prototype for evolutionary modification of fermentation. Fumarate reductase was later incorporated into the cytoplasmic membrane, and several electron carriers of suitable redox potential, including menaquinones, were gradually added to this system. After oxygen was accumulated in the biosphere, succinate dehydrogenase might originate by a modification of the fumarate reductase with the appearance of the higher potential quinones, ubiquinones. In most aerobic bacteria, ubiquinones remained as constituents of the respiratory chains with the concomitant loss of menaquinones. Then, in some aerobic or phototrophic bacteria, rhodoquinones appeared as electron carriers for maintaining redox balance under oxygen-limited or anaerobic conditions.

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