The Phenotypic and Chemotaxonomic Characteristics of Methanol-Utilizing Hyphomicrobium Strains Were Examined and Compared with Other Related Budding Bacteria

J. Gen. Appl. Microbiol., 33, 521-542 (1987)

CHARACTERIZATION AND IDENTIFICATION OF METHANOL-UTILIZING HYPHOMICROBI UM STRAINS AND A COMPARISON WITH SPECIES OF HYPHOMONAS AND RHODOMICROBI UM

TEIZI URAKAMI' ANDKAZUO KOMAGATA

Institute of Applied Microbiology, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan

(Received September 24, 1987)

The phenotypic and chemotaxonomic characteristics of methanol-utilizing Hyphomicrobium strains were examined and compared with other related budding bacteria. These Hyphomicrobium strains are facultatively methylotrophic, and utilize methanol, but not methane, by the serine pathway with formaldehyde incorporation. The strains were divided into two species, Hyphomicrobium vulgare and Hyphomicrobium methylovorum, based on DNA base composition, and the activities of catalase and urease. The major cellular fatty acid is straight-chain unsaturated C18:1 acid. The major ubiquinone is Q-9. Strains in other similar budding bacteria in the genera Hyphomonas, Pedomicrobium, Rhodomicrobium, and Hypho- microbium indicum do not utilize methanol, have different DNA base compositions, possess different cellular fatty acids, and have different quinone systems. Based on the above data, the methanol-utilizing Hyphomicrobium strains are clearly distinguished from the supposedly related genera.

The genus Hyphomicrobium includes facultatively methylotrophic, nonsporeforming, gram-negative, polarly flagellated, hyphal-budding, rod-shaped organisms that have a Q-9 ubiquinone system (1, 2), a cellular fatty acid composition consisting of a large amount of C18:1 acid (Type B)(], 3), and DNA base composition from 59.2 to 66.8 mol 0 guanine plus cytosine (G +C) (4). Hyphomicrobium strains were first isolated from enrichment cultures designed for autotrophic nitrifying bacteria, and had been regarded as an autotroph based on the

' Present address and address reprint requests to: Dr . T. Urakami, Niigata Research Laboratory, Mitsubishi Gas Chemical Co., Inc., Niigata 950-31, Japan. 521

522 URAKAMI and KOMAGATA VOL. 33 failure to use sugars for growth (5), the ability to oxidize formate and oxalate to CO2 and H2O with release of most of the energy as heat (6, 7), and the incorporation of 14C-labeled "C compounds" into cellular amino acids during growth in the presence of CO2 (8). However, LARGEet al. (9) reported that the ribulose diphos- phate cycle of C02-fixation did not function in Hyphomicrobium vulgare and autotrophy was unlikely. Furthermore, HIRSCHand CoNTI(10) reported that Hypho- microbium vulgare utilized primarily one-carbon compounds, such as methanol and methylamine. In Bergey's Manual of Determinative Bacteriology, 8th ed. (11), Hyphomicrobium vulgare was the only legitimate species, and Hyphomicrobium neptunium and Hyphomicrobium indicum were described as species incertae sidis. Hyphomicrobium vulgare utilized methanol, but the other two species did not (11). H. neptunium produced a single nonfilamentous prostheca (12), and metabolized exogenous amino acids (13). Recent studies of DNA-DNA homologies within Hyphomicrobium, Hyphomonas, Prosthecomicrobium, and Ancalomicrobium strains revealed that H. neptunium had a closer relationship to Hyphomonas polymorpha (14, 15). Based on this information, MOOREet al. (16) proposed the transfer of H. neptunium to the genus Hyphomonas, as Hyphomonas neptunium. Hyphomicrobium indicum which has 40 mol % G + C in DNA, differs from all other hyphomicrobia and related organisms with DNA base composition ranging from 59.2-66.8 mol° o G + C (11,16). This species also differs from the true-budding hyphal hyphomicrobia by the absence of buds and branching hyphae, and by its quite different metabolism. Therefore, it has been recommended that this organism be transferred to another genus (I1). During the course of taxonomic studies of methanol-utilizing bacteria, we divided them into 11 groups on the basis of morphological, physiological, and chemotaxonomical characteristics, and group 4 methanol-utilizing bacteria were placed in the genus Hyphomicrobium (17). IzuMi et al. (18) isolated a new species of obligate methylotrophic Hyphomicrobium, that was named Hyphomicrobium methylovorum. This name has since been validated (19). In the present study, we characterized the group 4 methanol-utilizing bacteria, and identified them as Hyphomicrobium vulgare and Hyphomicrobiumnmethylo- vorum. The strains in H. vulgare showed some characteristics different from the description of Hyphomicrobium (11), especially, in physiological characteristics. Therefore,we present a detailed recharacterization of the genus Hyphomicrobium, and of H. vulgare and H. methylovorum.

MATERIALS AND METHODS

Bacterial strains. The strains studied are listed in Table 1. In addition to the bacteria described previously (1, 24), bacteria closely related to this group were included in this study. H. vulgare TK 0401 T, and Hyphomicrobium sp. TK 0403 and TK 0404 were derived from the "B" strain originally isolated by MEVIUS(5) from 1987 Characterization and Identification of Hyphomicrobium 523

Elbe River water near Cuxhaven, Germany (10). Hyphomicrobium sp. TK 0405 was isolated using methanol as a carbon source (25). Hyphomicrobium sp. TK 0407 was derived from enrichment under an atmosphere containing CO (26). Hyphomicrobium sp. TK 0408, TK 0410, and TK 0411 were isolated by HIRSCHand CoNTI using methylamine as the carbon source (10). Hyphomicrobium indicum, Hyphomonas neptunium, Hyphomonas polymorpha, Hyphomonas oceanitis, Hyphomonas hirschiana, and Hyphomonas jannaschiana, and Rhodomicrobium vannielii strains were used as the reference. They did not utilize methanol, but all strains except H. indicum are prosthecate, budding bacteria (11,12,16, 27-29). The type strain is shown by the superscript "T" above the strain number. Identification methods. Group 4 methanol-utilizing bacteria were maintained on medium B (1). Strains of Hyphomicrobium indicum, Hyphomonas neptunium, H. oceanitis, H, hirschiana, and H, jannaschiana were maintained on Casitone-yeast extract medium containing 2 g of Casitone (Difco Laboratories, Detroit, Mich., U.S.A.), 1 g of yeast extract, 2 g of MgSO4.7H2O, and 1,000 ml of seawater; the pH of this medium was adjusted to 7.5. H. polymorpha strains were maintained on Hyphomonas medium (16) containing 2 g of Casitone, 1 g of yeast extract, 2 g of MgSO4.7HzO, and 1,000 ml of distilled water; the pH of this medium was adjusted to 7.5. An R. vannielii strain was maintained on medium B containing 0.5 g of ethanol instead of methanol. This strain was cultivated under anaerobic and photosynthetic conditions in screw-capped bottles completely filled with medium under illumination with an incandescent lamp (ca 2,OOOlux). Unless otherwise indicated, cultures were grown at 30 C. Cell shape, gram reaction, motility, and flagellar morphology were determined by using cells grown on medium B agar. Gram staining was done by the HUCKER-CoNNmodification (30). Motility was investigated by the hanging drop method, and flagellar morphology was determined by the staining method of TODA(3'1), and by using a preparation that was stained negatively with 2 % of phosphotungstic acid (pH 7.0), and an electron microscope. For comparison of the methanol-utilizing bacteria studied and other aerobic bacteria, the following characteristics were investigated in media containing methanol as substrate: Production of fluorescent pigments, nitrate reduction, methyl red reaction, Voges-Proskauer reaction, production of indole and hydrogen sulfide, hydrolysis of gelatin and starch, production of ammonia, nitrate respiration (anaerobic growth in the presence of nitrate), litmus milk reaction, oxidation- fermentation test (OF-test), nutritional requirement, utilization of nitrogen compounds, urease, oxidase, catalase, cultural temperature, cultural pH, and tolerance to sodium chloride (32). Utilization of carbon compounds was determined in liquid basal medium B after 3 weeks of cultivation. Acetate, formate, propionate, iso- butyrate, n-valeriate, lactate, succinate, oxalate, monomethylamine, dimethylamine, and trimethylamine were added at a concentration of 0.15%. L-Arabinose, D-xylose, D-glucose, D-mannose, D-fructose, D-galactose, maltose, sucrose, lactose, trehalose, D-sorbitol, D-mannitol, inositol, glycerol, soluble starch, ethanol, pectin, and methanol were added at concentrations of 0.5%. Utilization of methane and 524 URAKAMI and KOMAGATA VOL. 33

Table 1. Bacterial strains stud ied. 1987 Characterization and Identification of Hyphomicrobium 525

Table 1, (continued).

hydrogen was tested as described previously (32-34). Tolerance to sodium chloride. Tolerance to sodium chloride was tested in a basal medium containing 5 % sodium chloride. The basal medium used for H. indicum and Hyphomonas is Hyphomonas medium (16). Medium B, containing 0.5 g of ethanol instead of methanol was used for Rhodomicrobium; this strain was cultivated under anaerobic and photosynthetic conditions. Cultivations of bacteria. Cells cultivated for 3 days with shaking in a 1,000-ml conical flask containing 300 ml of medium were used for the analysis of G + C content in DNA, fatty acids, and the quinone system. Group 4 methanol-utilizing bacteria were cultivated in medium B broth. Hyphomicrobium indicum, Hyphomonas neptunium, H. oceanitis, H. hirschiana, and H. jannaschiana strains were cultivated in Casitone-yeast extract medium, H. polymorpha strains were grown in Hyphomonas medium, and an R. vannielii strain in medium B containing 0.5 g of ethanol instead of methanol. DNA base composition. DNA was extracted by the method of SAITOand MIURA(35), and G + C content in DNA was determined by reversed-phase high performance liquid chromatography as described previously (36). Cellular fatty acid composition. Cellular fatty acid composition and hydroxy fatty acid composition were determined as described previously (1,3,32-34). Quinone system and quinone homologues. Quinone systems were determined as described previously (1,2,32-34). 526 URAKAMI and KOMAGATA VOL. 33

RESULTS

Phenotypic characteristics o,f Hyphomicrobium and the group 4 bacteria Group 4 methanol-utilizing bacteria (Hyphomicrobium strains) were gram- negative, nonsporeforming, rod-shaped organism with pointed ends, oval-, egg-, or bean-shaped forms, 0.3-0.6pm in diameter, and 1.0-3.Opm in length. These bacteria produced mono- or bipolar filamentous outgrowths (hyphae) of varying length and 0.3-0.4pm in diameter when stained. The hyphae were not septate. Cells multiplied by budding hyphal tips. Mature buds became motile, broke off, and often attached themselves to a surface or to other cells to form clumps. Motility was lost soon after attachment. Electron micrographs of H. vulgare TK 0401 are shown in Fig. 1. Cells did not grow in nutrient broth or peptone broth. Colonies on

Fig. 1. Electron micrograph of Hyphomicrobium vulgare TK 0401T A: Swarm cell. B: "Mother" and developing "daughter" cells. Magnification bars represent 1 µm. 1987 Characterization and Identification of Hyphomicrobium 527 methanol-containing agar were shiny, smooth, raised, entire, white, and 1-2 mm in diameter after 3 to 6 days at 30°C. No water-soluble fluorescent pigments were produced by any of the strains. Most strains reduced nitrate, but Hyphomicrobium sp. TK 0410, and isolates TK 0417, TK 0422, TK 0424, TK 0425, and TK 0427 did not (Table 2). The methyl red test and Voges-Proskauer reaction were negative. Indole and hydrogen sulfide were not produced. Hydrolysis of gelatin and starch was negative. Ammonia was not produced. Nitrate respiration occurred in about 1/3 of strains (Table 2). Acid was not produced from sugars oxidatively or fermentatively. No vitamins or amino acids were essential for the growth of any strains. Ammonia and urea were utilized by all strains as a sole nitrogen source. Nitrate was utilized weakly by most strains. H, methylovorum TK 0412T, Hyphomicrobium sp. TK 0407, and isolate TK 0429 did not utilize nitrate (Table 2). About 1/2 of the strains produced urease (Table 2). All strains produced oxidase. Most strains produced catalase. H. vulgare TK 0401T and TK 0402, and Hyphomicrobium sp. TK 0403, TK 0404, TK 0405, and TK 0406 did not show catalase activity (Table 2). Good growth was observed between pH 6.0 and 8.0. All strains grew at 30°C, and did not grow at 47°C. Approximately 1/3 of the strains grew weakly at 42°C. The temperature range for growth differed among the strains (Table 2). None of the strains grew in the presence of 3° sodium chloride. All strains utilized methanol, monomethylamine, and pectin, but they did not utilize methane as a sole source of carbon. None of the strains utilized L-arabinose, D- xylose, D-glucose, D-fructose, D-galactose, maltose, sucrose, lactose, trehalose, D- sorbitol, D-mannitol, inositol, glycerol, soluble starch, propionate, iso-butyrate, n- valeriate, lactate, succinate, oxalate, or hydrogen. Some strains utilized acetate, formate, ethanol, dimethylamine, and trimethylamine (Table 2).

Tolerance to sodium chloride in Hyphomicrobium indicum, Hyphomonas strains, and Rhodomicrobium vannielii H. polymorpha and R. vannielii strains grew in a basal medium, but did not grow in the presence of 500 sodium chloride. On the other hand, Hyphomicrobium indicum, H. neptunium, H. oceanitis, H. hirschiana, and H. jannaschiana strains grew in the presence of 500 of sodium chloride, but did not grow in the basal medium. DNA base composition. The DNA base composition was determined for 11 strains in group 4 methanol-utilizing bacteria (Table 3), 6 strains of the genus Hyphomonas, and a strain of Hyphomicrobium indicum (Table 4). As shown in Table 3, the group 4 methanol-utilizing bacteria were divided into two subgroups. H. vulgare TK 0401T and TK 0402, and Hyphomicrobium sp. TK 0403, TK 0404, TK 0405, and TK 0406 had a DNA base composition ranging from 63.8 to 66.8 mol% G+C, and the other strains had DNA base composition ranging from 59.2 to 61.7 mol % G + C. On the other hand, 6 strains of the genus Hyphomonas had G + C contents ranging from 60 to 64mo1%, while Hyphomicrobium indicum TK 0433 had 44.2 mol 0 G + C (Table 4). Cellular fatty acid composition. All strains of group 4 methanol-utilizing 528 URAKAMI and KOMAGATA VOL. 33

Table 2. Biochemical and physiological characteristics

bacteria contained a large amount of straight-chain unsaturated C18:1 acid (Table 5). This cellular fatty acid composition is Type B (1). Small amounts of straight- chain saturated C16:0 acid, C18:0 acid and C19:0 acid, and cyclopropane C19.0 acid were also detected (Table 5). On the other hand, H. neptunium and H. polymorpha 1987 Characterization and Identification of Hyphomicrobium 529

strains contained a large amount of cyclopropane C17.0 acid, and H. oceanitis, H. hirschiana, and H. jannaschiana strains contained a large amount of straight-chain unsaturated C18 :1 acid. A Hyphomicrobium indicum strain contained a large amount of anteiso C15 : o acid (Table 6). 530 URAKAMI and KOMAGATA VOL. 33 1987 Characterization and Identification of Hyphomicrob ium 531 532 URAKAMI and KOMAGATA VOL. 33

Table 4. DNA base composition, quinone type, and DNA-DNA homology group of

I-11 strains oI group 4 meindnol-uullzlllg oaeieTIa eo1ILaiiieu 3-VI1 1.14:0 hydroxy acid and 3-OH C16:0 hydroxy acid (Table 5, (3)). But, these strains were divided into two subgroups on the basis of the ratio of 3-OH C14:0 to 3-OH C16:0 acid as shown in a previous report (3). On the other hand, H. neptunium and H. polymorpha contained 3-OH C11 :0 hydroxy acid, 3-OH C12.0 hydroxy acid, and 3- OH C13:0 hydroxy acid; H. oceanitis, H. hirschiana, and H. jannaschiana contained 3-OH C12:0 hydroxy acid and 3-OH C16:0 hydroxy acid; and R. vannielii contained 3-OH C16:0 hydroxy acid (Table 7). Quinone system. All strains of group 4 methanol-utilizing bacteria had Q-9 ubiquinone along with minor amounts of Q-8 and Q-10 (2) (Table 3). On the other hand, H. neptunium, H. polymorpha, H. oceanitis, and H. hirschiana strains had Q- 11 ubiquinone along with a considerable amount of Q-10 (approximately 1000 of the total ubiquinones) and minor amounts of Q-9 and Q-12 (Figs. 2 and 3); a H. jannaschiana strain had Q-10 ubiquinone along with minor amounts of Q-9 and Q- 11. A Hyphomicrobium indicum strain had the MK-7 menaquinone along with minor menaquinone MK-5, MK-6, and MK-8 components (Table 4).

DISCUSSION

The 32 strains of group 4 methanol-utilizing bacteria that we described (1) shared the same phenotypic characteristics except nitrate reduction, urease, catalase, nitrate respiration, utilization of nitrate, utilization of acetate, formate, ethanol, dimethylamine, and trimethylamine, and growth temperature. From the characteristics of group 4 methanol-utilizing bacteria, including H. vulgare, the type species of Hyphomicrobium, these group 4 methanol-utilizing bacteria seem to fit in the genus Hyphomicrobium. Furthermore, these bacteria share the same major cellular fatty acid composition and ubiquinone system (Tables 3 and 5). On the other hand, they could be divided into two subgroups on the basis of DNA base composition, 1987 Characterization and Identification of Hyphomicrobium 533

Hyphomicrobium indicum strain and the strains of the genera Hyphomonas and Rhodomicrobium.

and this division correlated with the activity of catalase and urease, and the ratio of 3-OH C14.0 hydroxy acid to 3-OH C16.0 hydroxy acid (3) (Table 8). Strains of subgroup 1 had DNA base composition ranging from 63.8 to 66.8 mol% G+C, and included H. vulgare TK 0401T (=Hirsch NQ 521) and TK 0402, and Hyphomicrobium sp. TK 0403 ( = Hirsch MEV 533-Gr), TK 0404 ( = Hirsch NQ 521- Gr), TK 0405 ( = Hirsch MC-630), and TK 0406 ( = Hirsch ZV-620). Subgroup 2 consisted of strains with DNA base composition ranging from 59.2 to 61.7 mol G+C, and included H. methylovorum TK 0412T, "Hyphomicrobium variable" TK 0413, Hyphomicrobium sp. TK 0407 (= Hirsch CO-558), TK 0408 (= Hirsch L-530), TK 0409 (= Tyler T37), TK 0410 (= Hirsch H-526), TK 0411(= Hirsch D-524), TK 0414, TK 0415, and TK 0416, and all strains isolated by us as methanol-utilizing bacteria. This grouping agrees well with the grouping based on DNA-DNA homology by MOOREand HIRSCH(14). GEBERSet al. (37) studied the level of genetic relatedness by DNA-DNA hybridization of 19 Hyphomicrobium strains which utilized one-carbon compounds and reported that these Hyphomicrobium strains could be divided into seven groups (A to G). Our subgroup 1 was equivalent to groups B and C of GEBERSet al., and our subgroup 2 contained their group E. However, these molecularly established groups did not correlate with nitrate respiration, utilization of nitrate, urease, growth temperature, and utilization of acetate, formate, ethanol, dimethylamine, and trimethylamine. The serine pathway of methanol metabolism was supported by a comparison of enzymes, as previously reported (24). Function of this system was suggested in H. vulgare (9), H. methylovorum KM 146T (=TK 0412) (18) and Hyphomicrobium sp. strain X (TK 0415) (38). These strains are included in group 4 methanol-utilizing bacteria. Complete tricarboxylic acid cycle was shown in Hyphomicrobium sp. strain X (TK 0415) (38). IZUMIet al, reported H. methylovorum as an obligate methylo- troph (18). But, several strains tested utilized acetate, formate, or ethanol as a sole carbon source, and these bacteria metabolized methanol by the serine pathway as 534 URAKAMI and KOMAGATA VOL. 33

Table 5. Cellular fatty acid composition

well as other facultatively methylotrophs (24, 33). Thus, we regard these bacteria as facultative methylotrophs. At the present time, we believe that Hyphomicrobium strains can be separated into two species that correlate with subgroups 1 and 2. Therefore, it seems adequate that subgroup 1 strains are identified as H, vulgare, and subgroup 2 bacteria as H. methylovorum. The characteristics of the genus Hyphomicrobium revealed in this study are 1987 Characterization and Identification of Hvphomicrobium 535 of the group 4 methanol-utilizing bacteria.

rather different from those described based on the original description (39) in Bergey's Manual of Determinative Bacteriology, 8th ed. (11), and such characteristics should be corrected according to the recent studies including the data in this study. Until now, budding and prosthecate bacteria (Hyphomicrobium, Hyphomonas, Pedomicrobium, and Rhodomicrobium strains) have been discussed based on morphology, physiology, and chemotaxonomy (11-16,26-29,37,40-46). MOORE et 536 URAKAMI and KOMAGATA VOL. 33

Table 6. Cellular fatty acid composition of Hyph omicrobium indicum

°,~ of total

al. (16) proposed the transfer of Hyphomicrobium neptunium to the genus Hyphomonas, as Hyphomonas neptunium. The transfer of Hyphomicrobium indicum to another genus has been recommended on the basis of DNA base composition, and morphological and physiological characteristics by HIRscH (11). The data for the cellular fatty acid composition and the quinone systems in the strains of Hyphomicrobium indicum, and of the species of Hyphomonas and Rhodomicrobium genera investigated in this study and a previous study (47) have a fairly good correlation with these rearrangements (Table 8). Therefore, it seems likely that Hyphomicrobium indicum falls into a different taxon based on low DNA base composition, cellular fatty acid composition of anteiso C15.0 acid, and menaquinone system, and it should be separated from Hyphomicrobium. Hyphomonas strains are divided into three groups based on the cellular fatty acid composition and the quinone systems. H. neptunium and H. polymorpha strains contain a large amount of cyclopropane C17.0 acid and ubiquinone Q-11 system, H. oceanitis and H. hirschiana strains contain a large amount of straight-chain unsaturated CI8.1 acid and ubiquinone Q-11 system. And H. jannaschiana strains contain a large amount of straight-chain unsaturated C18.1 acid and ubiquinone Q-10 system. Hyphomonas strains except H, jannaschiana are unique in having a large amount of cyclopropane C17.0 acid and/or ubiquinone Q-11.

Description of Hyphomicrobium Stutzer and Hartleb 1899 (Approved Lists 1980) Cells are gram-negarive, non-sporeforming, rod-shaped with pointed ends, oval-, egg-, or bean-shaped forms, 0.3-0.6 pm in diameter, and 1.0-3.0 pm in length, and produce mono- or bipolar filamentous outgrowths (phyhae) of varying length 0.3-0.4pm in diameter when stained. The hyphae are not septate. Cells multiply by budding at the tips of the hyphae. Mature buds become motile, break of, and often 1987 Characterization and Identification of Hyphomicrobium 537

Table 7. Percentage hydroxy acid composition of the strains of the genera Hyphomonas and Rhodomicrobium.

attach themselves to surface or other cells to form clumps. Motility is lost soon after attachment. Cells do not grow in nutrient broth or peptone broth. Colonies on methanol-containing agar are shiny, smooth, raised, entire, white, and 1-2 mm in diameter after 3 to 6 days at 30°C. A water-soluble fluorescent pigment is not produced on King A and King B media containing methanol. Methyl red and Voges-Proskauer tests are negative. Indole and hydrogen sulfide are not produced. Hydrolysis of gelatin and starch is not observed. Ammonia is not produced. Acid is not produced from sugars, oxidatively or fermentatively. Methanol, monomethylamine, and pectin are utilized as a sole source of carbon, but methane is not utilized. L-Arabinose, D-xylose, D-glucose, D- mannose, galactose, maltose, sucrose, lactose, trehalose, D-sorbitol, D-mannitol, inositol, glycerol, soluble starch, propionic acid, iso-butyric acid, n-valeric acid, 538 URAKAMI and KOMAGATA VOL. 33

Fig. 2. High-performance liquid chromatogram of ubiquinones isolated from cells of Hyphomonas polymorpha TK 0435TT lactic acid, succinic acid, and oxalic acid are not utilized. Utilization of acetic acid, formic acid, and ethanol differs among the strains. Vitamins and amino acids are not required for growth. Ammonia and urea are utilized as a sole source of nitrogen. Oxidase is produced. Aerobic. Metabolism is strictly respiratory and not fermentative. Good growth occurs between pH 6.0 and 8.0. Good growth occurs at 30°C. Growth does not occur in media containing 3 sodium chloride. DNA base composition ranges from 59.2 to 66.8 mol 0 guanine plus cytosin. The cellular fatty acids are composed of a large amount of straight- chain unsaturated C18.1 acid, and a small amount of 3-OH C14:0 hydroxy acid, and 3-OH C16:0 hydroxy acid. The major ubiquinone is Q-9. Methanol is utilized by the serine pathway with formaldehyde incorporation. The type species is H, vulgare Stutzer and Hartleb 1899. The differential characteristics of H. vulgare, H. methylovorum, and other genera of prosthecate budding bacteria are shown in Table 8. A description of H. vulgare and H. methylovorum are as follows:

Hyphomicrobium vulgare Stutzer and Hartleb 1899 Nitrate is reduced to nitrite. Nitrate respiration (anaerobic growth in the presence of nitrate) is observed. Methanol, monomethylamine, and pectin are utilized as a sole source of carbon, but methane is not utilized. Ammonia, nitrate, and urea are utilized as a sole source of nitrogen. Oxidase is produced, but urease and catalase are not produced. The DNA base composition is 64.3-66.8 mol% 1987 Characterization and Identification of Hyphomicrobium 539

Fig. 3. Mass spectrum of ubiquinone isolated from the cells of Hyphomonas polymorpha TK 0435T. This ubiquinone preparation exhibited intense fragment peaks at m/z 197 and 235. guanine, plus cytosine. The type strain is TK 0401 (= NCIB 9698 = Mevius strain B = Hirsch NQ 521). Strains TK 0401 to TK 0406 were reidentified as H. vulgare.

Hyphomicrobium methylovorum Izumi, Takizawa, Tani, and Yamada 1982 Almost strains reduced nitrate to nitrite (Some strains did not reduce nitrate to nitrite). Hardly any strains show nitrate respiration, but some strains do. Methanol, monomethylamine, or pectin is utilized as sole sources of carbon, but methane is not utilized. Ammonia and urea are utilized as a sole source of nitrogen. Nearly all strains utilize nitrate, but some strains do not. Oxidase and catalase are produced. Almost all strains produce urease, but some strains do not. The DNA base 540 URAKAMI and KOMAGATA VOL. 33

Table 8. Characteristics of the species and the

composition is 59.3 to 61.7 mol% guanine plus cytosine. The type strain is TK 0412 (= IFO 14180 = Izumi et al. KM 146), which was isolated by Izumi et al. in 1982. Strains TK 0407 to TK 0431 and TK 0441 were reidentified as H. methylovorum.

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