INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Jan. 1992, p. 84-92 Vol. 42, No. 1 0020-771319210 10084-09$02.00/0 Copyright 0 1992, International Union of Microbiological Societies Transfer of Pseudomonas aminovorans (den Dooren de Jong 1926) to Aminobacter gen. nov. as Arninobacter aminovorans comb. nov. and Description of Aminobacter aganoensis sp. nov. and Aminobactev niigataensis sp. nov. TEIZI URAKAMI,l* HISAYA ARAKI,2 HIROMI OYANAGI,* KEN-ICHIRO SUZUKI,3 AND KAZUO KOMAGATA4 Biochemicals Division, Mitsubishi Gas Chemical Co., Seavans-N Building, 1-2-1, Shibaura, Minato-ku, Tokyo 105,' Niigata Research Laboratory, Mitsubishi Gas Chemical Co., Niigata 950-31 ,2 Japan Collection of Microorganisms, The Institute of Physical and Chemical Research, Wacho-shi, Saitama 351-01,3 and Institute of Applied Microbiology, The University of Tokyo, Bunkyo-ku, Tokyo I 13,4Japan We compared non-methane-, non-methanol-, and methylamine-utilizing bacteria, including Pseudomonas aminovorans, a tetramethylammonium-utilizing bacterium, and an N,N-dimethylformamide-utilizingbacte- rium. These bacteria are gram-negative, nonsporeforming, subpolarly flagellated, rod-shaped organisms. Reproduction occurs by budding. The DNA base compositions are 62 to 64 mol% guanine plus cytosine. The cellular fatty acids contain a large amount of acid. The major hydroxy acid is 3-OH C1z:oacid. The major ubiquinone is ubiquinone Q-lo. These bacteria are clearly separated from authentic Pseudomonas species (Pseudornonas Jiuorescens rRNA group) on the basis of utilization of methylamine, morphological and chemotaxonomic characteristics, DNA-DNA homology, and rRNA-DNA homology. They were divided into three subgroups on the basis of their physiological characteristics and DNA-DNA homology data. A new genus, Aminobacter, and three new species, Aminobacter aminovorans comb. nov., Aminobacter aganoensis sp. nov., and Aminobacter niigataensis sp. nov., are proposed. The type species of Aminobacter gen. nov. is A. aminovorans, with type strain JCM 7852. The type strain of A. aganoensis is strain TH-3 (= JCM 7854), and the type strain of A. niigataensis is strain DM-81 (= JCM 7853). Although a large number of methane- and methylamine- philus (41, 42), and Paracoccus aminovorans (41, 42) were utilizing bacteria (and a large number of methanol- and described. Strains TH-3T and DM-81T were non-methane-, methylamine-utilizing bacteria have been described (23, 29, non-methanol-, monomethylamine-, dimethylamine-, and 43-54), only a few non-methane-, non-methanol-, and meth- trimethylamine-utilizing, nonsporeforming, gram-negative, ylamine-utilizing have been found to date (4, 10, 13, 22, 29, subpolar1y flagellated, rod-shaped organisms with a ubiqui- 32, 4042). Pseudomonas aminovorans (4), Pseudomonas none Q-10 system, a cellular fatty acid composition which sp. strain MA (32), and Pseudomonas sp. strain MS (22) included a large amount of C18:' acid, a hydroxy fatty acid have been reported to be gram-negative methylamine-utiliz- composition which included a large amount of 3-hydroxy ing bacteria. These three bacteria were distinguished from C12:* acid, and a DNA base composition ranging from 62 to methane- and met hanol-utilizing bacteria on the basis of 64 mol% guanine plus cytosine (G+C) (40, 42). These their failure to utilize methanol and methane. De Ley et al. characteristics were identical for Pseudomonas amino- (5) studied the rRNA cistron similarities of Pseudomonas vorans JCM 7852T, Pseudomonas sp. strain MA, and Pseu- and Pseudomonas-like strains and reported that the type domonas sp. strain MS, as reported previously (47, 48). strain of P. aminovorans was actually a member of the In this study, we characterized five methylamine-utilizing Rhizobium loti rRNA branch and not a member of the genus bacteria, including strains TH-3T and DM-81T, and clarified Pseudomonas. Furthermore, Green and Gillis (12) observed the differentiation of these organisms from authentic Pseu- the budding multiplication of P. aminovorans strains and domonas species; in this paper we propose the transfer of discussed the relationship of these organisms to R. loti and Pseudomonas aminovorans to a new genus, Aminobacter, Blastobacter species. However, these authors did not make as Aminobacter aminovorans and the establishment of two formal taxonomic proposals concerning Pseudomonas ami- new species, Aminobacter aganoensis and Aminobacter novorans. Recently, Tsuji et al. (39) described the results niigataensis. of a 16s rRNA sequence analysis performed to determine the phylogenetic relationships among methylotrophs; in this analysis they included methanol- and methylamine-utilizing MATERIALS AND METHODS Methylophilus, Methylobacterium, and Hyphomicrobium strains, but non-methane-, non-methanol-, and methyl- Bacterial strains. The strains which we studied are listed in amine-utilizing bacteria were not studied. Table 1. Strain TH-3T was isolated as a TMAH-utilizing Previously, we described the biodegradation of tetrameth- bacterium, and strain DM-81T was isolated as a DMF- ylammonium hydroxide (TMAH) and N,N-dimethylforma- utilizing bacterium (40, 42). Pseudomonas aminovorans mide (DMF) by bacteria (40, 42); in this work strains TH-3T JCM 7852T (= NCIB 9039T = NCTC 10684T = ATCC 23314T) (4), Pseudomonas sp. strain ATCC 23819 (= strain (T = type strain), DM-81T, and TH-15, Paracoccus amino- MA = NCIB 11590) (32), and Pseudomonas sp. strain ATCC 25262 (= strain MS = NCIB 11591) (22) are non-methane-, non-methanol-, and methylamine-utilizing bacteria (47, 48). * Corresponding author. Pseudomonas diminuta JCM 2788T (= ATCC 11568T = 84 VOL.42, 1992 AMINOBACTER GEN. NOV. 85 TABLE 1. Differentiating physiological characteristics of methylamine-utilizing bacteria Utilization of: Production Nitrate Growth in of acid from Strain reduction Dimethylamine Formamide ~~~~~.$DMF TMAH ~~~~~s,” Pseudomonas - + - aminovorans JCM 78XT Pseudomonas sp. strain - + - ATCC 23819 Pseudomonas sp. strain - + - ATCC 25262 TH-3T Wa w or - + DM-81T W w or - + + + - a w, weak. IAM 1513T), Pseudomonas vesicularis IF0 12165T (= acid composition and hydroxy fatty acid composition were ATCC 11426T = IAM 12105T), Sphingomonas paucimobilis determined as described previously (49). (Pseudomonas paucimobilis [17, 591) IF0 13935T (= ATCC Quinone and quinone homologs. Cells of reference strains 29837T = NCTC 11030T), Sphingomonas paucimobilis (Fla- that were cultivated aerobically for 2 days were used for the vobacterium devorans [17, 591) NCIB 8195 (= ATCC 10829 quinone analysis. Quinone systems were determined as = DSM 30198 = CCM 72), Sphingomonas capsulata (Fla- described previously (47). In the abbreviations used for vobacterium capsulatum [17, 591) NCIB 9890T (= ATCC ubiquinones, Q indicates ubiquinone, and the number indi- 14666T = IF0 12533T), Pseudomonas aeruginosa JCM cates the number of isoprenoid units in the side chain (e.g., 5962T (= ATCC 10145T = NCIB 8295T = NCTC 10332T = Q-9, Q-lo, etc.). NRRL B-771T), and Pseudomonas jluorescens NCIB 9046T DNA base composition. Cells that were cultivated aerobi- (= ATCC 13525T = CCM 21FiT = DSM 50090T = JCM cally for 2 days were used for the DNA analysis. DNA was 5963T) were used as reference strains. Strains TH-3T and extracted by using the method of Saito and Miura (31), and DM-81T and the three methylamine-utilizing bacteria resem- the G+C content was determined by reversed-phase high- bled Pseudomonas diminuta , Pseudomonas vesicularis , S. performance liquid chromatography as described by paucimobilis, and S. capsulata on the basis of their cellular Tamaoka and Komagata (36). fatty acid compositions and quinone systems (16, 25, 26, 58, DNA-DNA hybridization. DNA-DNA hybridization was 59). Pseudomonas aeruginosa and Pseudomonas Jzuo- carried out at 68°C by using the method of Kaneko et al. (21). rescens are typical species of the genus Pseudomonas. The The DNAs of strains TH-3T and DM-81T, Pseudomonas bacterial strains were maintained on PYG agar (pH 7.0) (41). aminovorans JCM 7852T, Pseudomonas diminuta JCM Identification methods. PYG agar was used for precultur- 27MT, and S. paucimobilis IF0 13935T were labeled with ing and as the basal medium for strains TH-3T and DM-81T, [1‘,2’, 5-3H]dCTP by the nick translation method, using a Pseudomonas aminovorans, Pseudomonas sp. strain ATCC type TRK 7000 kit (Amersham International plc, Amersham, 23819, and Pseudomonas sp. strain ATCC 25262. Unless United Kingdom). otherwise stated, the strains were cultivated aerobically in PYG broth at 30°C. Cell form, Gram reaction, motility, and RESULTS multiplication were determined by using cells grown on PYG agar. Motility was observed by using the hanging drop Phenotypic characteristics of methylamine-utilizing bacte- method. Flagellar morphology and multiplication were de- ria. The methylamine-utilizing bacteria (strains TH-3T and termined by the staining method of Toda (37), using a light DM-81T, Pseudomonas aminovorans JCM 7852T, Pseudo- microscope, and by using an electron microscope when the monas sp. strain ATCC 23819, and Pseudomonas sp. strain organisms were negatively stained with 2% phosphotungstic ATCC 25262) were gram-negative, nonsporeforming, rod- acid (pH 7.0). Granules of poly-P-hydroxybutyrate in cells shaped, methylamine-utilizing organisms that were 0.5 to 0.9 cultivated in medium containing 0.5% (wt/vol) DL-P-hy- by 1.0 to 3.0 pm and had rounded ends. Nonmucoid colonies droxy-n-butyric acid (sodium salt) were stained by using the were formed on PYG, nutrient, or methylamine-containing