INTERNATIONAL JOURNALOF SYSTEMATIC BACTERIOLOGY,OCt. 1995, p. 863-867 Vol. 45. No. 4 0020-77 13/95/$04.00+ 0 Copyright 0 1995, International Union of Microbiological Societies Characteristics of Newly Isolated Xanthobacter Strains and Fatty Acid Compositions and Quinone Systems in Yellow-Pigmented Hydrogen-Oxidizing Bacteria TEIZI URAKAMI,’* HISAYA ARAKI,2 AND KAZUO KOMAGATA3-(- Biochemicals Division, Mitsubishi Gas Chemical Co., Marunouchi, Chiyoda-ku, Tokyo 100, ‘ Niigata Research LaboratoT, Mitsubishi Gas Chemical Co., Tayuhama, Niigata 950-31, and Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo 113,3Japan Newly isolated Xanthobacter strains were characterized. In addition, the fatty acid compositions and quinone systems of gram-negative, yellow-pigmented, hydrogen-oxidizing bacteria belonging to the genera Xanthobacter, Hydrogenophaga, and Variovorax and related species were studied. Xanthobacter strains are nitrogen-fixing organisms that have a Q-10 ubiquinone system; the cellular fatty acids of these organisms include high levels of Cls,l acid, and their hydroxy fatty acids include high levels of 3-OH c16:o acid. Hydrogenophaga strains are polarly flagellated organisms that have a Q-S ubiquinone system. These bacteria can be divided into two groups on the basis of cellular fatty acid and hydroxy fatty acid compositions. Variovorax strains are peritrichously flagellated, non-nitrogen-fixing organisms that have a Q-S ubiquinone system; the cellular fatty acids of these strains include high levels of C16:o,c16:1 and C18:. acids, and their hydroxy fatty acids include 3-OH CIo:oand 2-OH C14:oacids. Xanthobacter, Hydrogenophaga, and Variovorax strains can be clearly distinguished from each other on the basis of their quinone systems and cellular fatty acid compositions. Previously (28-31), we described a group of gram-negative and Xanthobacterflavus by Wiegel et al. (38) and Malik and methanol-utilizing bacteria on the basis of their morphological Claus (22), respectively. In 1987, Xanthobacter agilis (1 8) was characteristics, utilization of carbon compounds, DNA base described as a third Xanthobacter species, and this species compositions, DNA-DNA hybridization data, cellular fatty could be clearly distinguished from the two other Xanthobacter acid compositions, ubiquinone systems. and enzyme electro- species on the basis of pleomorphism, motility, and colony phoretic properties. The group 5 bacteria that we described color (2). X autotrophicus DSM 432T (T = type strain), DSM (29, 30) are nitrogen-fixing, hydrogen-oxidizing, non-spore- 431, DSM 685, DSM 1393 (= JCM 786l), DSM 1618 (= JCM forming, gram-negative, rod-shaped, yellow-pigmented organ- 7862), DSM 2009 (= JCM 7863), and DSM 597 (= JCM 7864), isms that have a Q-10 ubiquinone system and cellular fatty X jlavus NCIB 1007T (= DSM 338‘), and all of our isolates acids that include high levels of Xanthobacter strains (22, (strains BY-1, BY-3 to BY-11, and BY-15) were gram-nega- 35, 38) are included in this group. tive, non-spore-forming, rod-shaped, methanol-utilizing organ- Coiynebacterium autotrophicum (6) and “Mycobacteriumjla- isms whose cells were 0.5 to 0.9 by 1.0 to 3.0 pm and had vum” (= “Microbacteriumflavum”) (14, 23) were transferred rounded ends. Nonmucoid yellow colonies were produced on to the new genus Xanthobacter as Xanthobacter autotrophicus PYG medium, nutrient medium, or methanol-containing me- TABLE 1. Quinone compositions of yellow colony-forming bacteria % of total quinones Ubiquinone homologs Menaquinone homologs Strain MK-A MK-7 MK-8 M K-9 MK-10 Q-7 Q-8 Q-9 Q-10 ____ HO H2 HO H2 Ho H, H,, HZ H() FI2 Colynebactenurn flavescens NCIB 8707T 1.6 0.9 9.3 11.3 14.7 58.4 0.4 3.5 “Mycobactenurn flavurn subsp. rnethanicurn” 1.0 0.2 2.3 9.8 0.3 86.4 0.1 NCIB 9738 “Mycobacterium flavum subsp. methanicum” 0.8 2.3 10.4 86.5 NCIB 9742 Hydrogenophaga flava DSM 619r 0.3 84.2 15.4 0.1 Hydrogenophaga pseudoflava DSM 1034T 1.3 94.2 4.5 Hydrogenophaga palleronii DSM 63T 1.4 96.6 1.9 0.1 Variovoraxparadoxus DSM 30034T 0.9 97.9 1.2 * Corresponding author. Mailing address: Biochemicals Division, Mitsubishi Gas Chemical Co., Mitsubishi Building, Marunouchi, Chiyoda-ku, Tokyo 100, Japan. Phone: (03) 3283-4833. Fax: (03) 3283- 5184. j- Present address: Department of Agricultural Chemistry, Tokyo University of Agriculture, Sakuragaoka 1-1-1, Setagaya-ku, Tokyo 156, Japan. 863 864 NOTES INT.J. SYST. BACTERIOL. TABLE 2. Cellular fatty acid compositions of yellow colony-forming bacteria % of cyclo- 10-Methy1 3-Hydroxy Straight-chain acids propane acids acids Strain acids 2-OH ~~~ ~~~ Corynebacterium flavescens NCIB 8707T 9.0 0.9 7.4 74.4 0.3 8.0 “Mycobacteriumflavum subsp. methanicum” 0.1 7.2 25.5 6.7 0.7 4.5 45.3 0.2 0.5 2.8 2.9 3.6 NCIB 9738 “Mycobacterium flavum subsp. methanicum” 0.1 8.1 26.7 7.7 0.7 6.0 42.0 0.2 3.5 1.9 3.1 NCIB 9742 Hydrogenophaga flava DSM 619T 3.3 0.8 32.4 56.6 0.3 0.3 2.8 0.5 3.0 Hydrogenophagapseudoflava DSM 1034T 2.2 0.5 32.2 52.7 0.4 0.4 10.4 0.3 1.2 Hydrogenophaga palleronii DSM 63T 0.2 0.1 31.5 45.2 1.0 0.2 21.4 0.1 0.2 Variovorax paradoxus DSM 30034T 0.3 0.9 33.9 34.3 1.1 0.4 17.2 3.9 2.1 5.3 dia. The cells occurred singly or rarely in pairs and were non- our isolates did not require amino acids for growth. The re- motile. The organisms grew abundantly in nutrient broth and quirement for vitamins varied among strains. X. autotrophicus PYG broth. No water-soluble fluorescent pigment was pro- DSM 432T, DSM 431, DSM 685, DSM 1393, DSM 1618, and duced on King B medium. Granules of poly-(3-hydroxybutyric DSM 2009 did not require vitamins for growth, but X flavus acid accumulated in the cells. The methyl red and the Voges- NCIB 10071T,X. autotrophicus DSM 597, and all of our iso- Proskauer tests in glucose phosphate broth were negative. In- lates had an absolute requirement for biotin. On the basis of dole and hydrogen sulfide were not produced. Hydrolysis of these physiological characteristics, all of our isolates were iden- gelatin and starch was not observed. Ammonia was produced. tified as X. flavus strains, and strain DSM 597 was reidentified Denitrification was negative. Litmus milk was not changed. All as a member of X. jlavus. of the strains utilized glycerol, succinic acid, citric acid, acetic The yellow-pigmented bacteria Colynebacterium flavescens acid, propionic acid, ethanol, Nfl-dimethylformamide, and (= Microbacterium flavum’,) (5, 8, 14) and “Mycobacterium hydrogen, but did not grow at the expense of L-arabinose, flavum subsp. methanicum” (20) were confused with X flavus galactose, maltose, lactose, trehalose, D-glucitol, D-mannitol, (= “Mycrobacteriumflavum’, 301T [ = NCIB 1007T]) until X inositol, soluble starch, cellobiose, L-histidine, L-phenylalanine, flavus and C. flavescens were described by Malik and Claus (22) tetramethylammonium hydroxide, or methane. All strains ex- and Barksdale et al. (5), respectively. C. flavescens (5) and cept X. autotrophicus DSM 431 utilized methanol; all strains “Mycobacteriumflavum subsp. methanicum” strains are gram- except X. autotrophicus DSM 431 and DSM 597 utilized D- positive bacteria and do not utilize hydrogen, methanol, glucose; and all strains except X autotrophicus DSM 431, DSM monomethylamine, dimethylamine, trimethylamine, tetrameth- 1618, and DSM 2009 utilized malonic acid. Utilization of D- ylarnmonium hydroxide, and NJV-dimethylformamide. Fur- xylose, D-mannose, D-fructose, sucrose, D-ribose, monomethyl- thermore, these organisms have different menaquinone sys- amine, dimethylamine, and trimethylamine varied among the tems, as shown in Table 1. C. flavescens contains mycolic acid strains. None of the strains produced acid oxidatively from and a high level of CIS:, acid, whereas “Mycobacteriurnflavum L-arabinose, D-xylose, D-glucose, D-mannose, galactose, mal- subsp. methanicum” strains contain mycolic acid, a high level tose, lactose, trehalose, D-glucitol, D-mannitol, inositol, glyc- of C18:1acid, and a relatively high level of C1(,:oacid, as shown erol, and soluble starch. X. autotrophicus DSM 432T, DSM 431, in Tables 2 and 3. Hydroxy fatty acids have not been detected and DSM 68.5 produced acid weakly from D-fructose, and X. in these strains. autotrophicus DSM 1393, DSM 1618, and DSM 2009 produced The yellow-pigmented hydrogen-oxidizing bacteria (2) acid weakly from D-fructose and sucrose. However, other Pseudomonas flava , Pseudomonas pseudoflava , Pseudomonas strains did not produce acid from D-fructose and sucrose. Acids palleronii, and Pseudomonas taeniuspiralis were placed in the were not produced fermentatively. Xanthobacter strains and new genus Hydrogenophaga as Hydrogenophaga flava, Hydro- TABLE 3. Hydroxy fatty acid compositions presence and carotenoid pigments of yellow colony-forming bacteria and the presence of mycolic acids in these bacteria 3-Hydroxy acid composition (%) 2-Hydroxy acid Presence of Carotenoid pigment Strain composition absorption 3-OH 3-OH 3-OH (% of 2-OH C14:o) acids maximum (nm) clUfl c15:0 c16:o Corynebacterium flavescens NCIB 8707T + 450 “Mycobacterium fluvum subsp. methanicum” NCIB 9738 + 450 “Mycobacterium fluvum subsp. methanicum” NCIB 9742 + 450 Hydrogenophaga flava DSM 619T 100 - 450 Hydrogenophaga pseudoflava DSM 1034T 100 - 450 Hydrogenophaga palleronii DSM 63T 20.7 79.3 - 450 Variovorax paradoxus DSM 30034T 100 100 - 420 VOL.45, 1995 NOTES genophaga pseudoflava, Hydrogenophaga palleronii, and Hydro- genophaga taeniospiralis, respectively, by Willems et al. in 1989 (39). Willems et al. (40) later described another new genus, the genus Variovorax, for Alcaligenes paradoxus, which became Variovorax paradoxus. These bacteria oxidize hydrogen, but do not utilize methanol, monomethylamine, dimethylamine, tri- methylamine, tetramethylammonium hydroxide, and Nfl-di- methylformamide.