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Mycoplana Ramosa and Mycoplana Segnis INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Oct. 1990, p. 43U2 Vol. 40, No. 4 0020-7713/90/040434-09$02.00/0 Copyright 0 1990, International Union of Microbiological Societies Recharacterization and Emended Description of the Genus Mycoplana and Description of Two New Species, Mycoplana ramosa and Mycoplana segnis TEIZI URAKAMI,l* HIROMI OYANAG1,l HISAYA ARAK1,l KEN-ICHIRO SUZUKI,’ AND KAZUO KOMAGATA3 Niigata Research Laboratory, Mitsubishi Gas Chemical Co., Tayuhama, Niigata 950-31I; Japan Collection of Microorganisms, The Institute of Physical and Chemical Research, Wako-shi, Saitama 351 -012; and Institute of Applied Microbiology, The University of Tokyo, Bunkyo-ku, Tokyo 113,3 Japan The phenotypic and chemotaxonomic characteristics of Mycuplana strains were examined. These strains were gram-negative, peritrichously flagellated, branching, filament-forming bacteria. The cellular fatty acids consisted of large amounts of straight-chain unsaturated ClSz1acid, as well as straight-chain saturated C16:o acid and unsaturated ClGZ1acid. The major ubiquinone was ubiquinone Q-lo. The Mycoplana strains were divided into four groups, including one group containing Mycuplana dimurpha ATCC 4279T (= IF0 13291T = NCIB 9439T) (T = type strain) and another group containing Mycuplana bullata ATCC 427tlT (= IF0 13290T = NCIB 9440T), on the basis of DNA base composition, major hydroxy fatty acid composition, mucosity of colonies, acid production from sugars, and utilization of carbon compounds. This division into groups was supported by the results of DNA-DNA homology experiments. Two new species, Mycoplana rumusa and Mycoplana segnis, are proposed. The type strain of M. ramusa is strain TKO053 (= NCIB 9440), and the type strain of M. segnis is strain TKO059 (= IF0 13240 = ATCC 21756). The genus Mycoplana was first defined by Gray and MATERIALS AND METHODS Thornton in 1928 (6) as a group of soil bacteria with Bacterial strains. The sources and reference collection branching cells and the ability to decompose aromatic com- numbers of the strains which we studied are shown in Table pounds. These authors described two species, Mycoplana 1. Names which do not appear on the Approved Lists of dimorpha (the type species) and Mycoplana bullata, and Bacterial Names (13) are enclosed in quotation marks. Since placed them in the genus Mycoplana in the family Mycobac- few strains of Mycoplana are available, we examined only teriaceae. The Mycoplana species were subsequently placed the following strains from established collections: M. bullata in the order Actinomycetales on the basis of morphological TK0051T (= ATCC 4278T) (T = type strain), TK0052T (= characteristics by several workers (5, 9, 10, 14). In particu- IF0 13290T), and TK0053T (= NCIB 9440T) and M. dimor- lar, Sukapure et al. (14) suggested that Mycoplana strains pha TKOOST (= ATCC 4279T), TK0056T (= IF0 13291T), are related to members of the genus Oerskovia morphologi- and TK0057T (= NCIB 9439T). Strains TKO065 and TKO066 cally. The different editions of Bergey’s Manual of Determi- were isolated by us. M. dimorpha TKO058 (= IF0 13213) native Bacteriology placed the genus Mycoplana in the was isolated as a cephalosporin-producing bacterium (T. family Mycobacteriaceae or in the family Pseudomona- Takahashi, Y. Yamazaki, K. Kato, and M. Isono, U.S. daceae (2) or left it out completely (3). In Bergey’s Manual patent 3,816,253, June 1974), and Mycoplana sp. strain of Systematic Bacteriology (ll), the genus Mycoplana was TK0059T (= IF0 13240T) was isolated as a 7-amino-3- mentioned only in the description of the genus Oerskovia. methylcephem compound-producing bacterium (T. Taka- However, the genus Mycoplana and M. dimorpha and M. hashi and K. Kamahara, U.S. patent 3,749,641, July 1973). bullata appear on the Approved Lists of Bacterial Names All strains were maintained on PYG agar containing 0.5% (13). A pink-pigmented facultatively methylotrophic bacte- (wthol) peptone, 0.5% (wt/vol) yeast extract, 0.5% (wthol) rium, “Mycoplana rubra” NCIB 10409, was deposited in the glucose, and 2.0% (wthol) agar; this medium was adjusted to National Collection of Industrial Bacteria (Aberdeen, United pH 7.0 with 1 M NaOH. Kingdom) by M. R. Rhodes. This strain was included in Morphological, biochemical, and physiological characteri- zation. Unless otherwise stated, the strains were cultivated group 2 of the methanol-utilizing bacteria that we described at 30°C. Cell form, Gram reaction, and motility were deter- previously (16). We investigated the previously described mined after cells were grown on PYG agar. Motility was Mycoplana strains and compared them with “M. rubra.” investigated by using the hanging drop method, and flagellar In this paper we present a detailed recharacterization of morphology was determined by electron microscopy, using a M. dimorpha and M. bullata and propose two new species, preparation that was negatively stained with 2% phospho- Mycoplana ramosa and Mycoplana segnis. Furthermore, we tungstic acid (pH 7.0). Granules of poly-P-hydroxybutyrate discuss the relationship of the genus Mycoplana to the in cells cultivated in PYG medium containing 0.5% (wt/vol) genera Oerskovia, Listeria, and Arthrobacter. DL-(3-hydroxy-n-butyric acid (sodium salt) were stained by using a method that has been described previously (16). Biochemical and physiological characteristics were investi- gated by using previously described methods (18). Vitamin requirements were assayed in liquid basal medium B (18) * Corresponding author. with the methanol replaced by 1% Casamino Acids (vitamin 434 VOL.40, 1990 NEW MYCOPLANA SPECIES 435 TABLE 1. Bacterial strains studied ~~ Species or isolate Strain Other designation(s)<’ Sourcea Reference M. bullata TK0051T ATCC 4278T, IF0 13290T, NCIB 9440T ATCC M. bullata TK0052T ATCC 4278T, IF0 13290T, NCIB 9440T IF0 M. bullata TK0053T ATCC 4278T, IF0 13290T, NCIB 9440T NCIB M. dimorpha TKOOST ATCC 4279*, IF0 13291T, NCIB 9439T ATCC M. dimorpha TK0056T ATCC 4279T, IF0 13291T, NCIB 9439T IF0 M. dimorpha TK0057T ATCC 4279T, IF0 13291T, NCIB 9439T NCIB M. dimorpha TKO058 ATCC 21759, IF0 13213 IF0 Takahashi et al.b Mycoplana sp. TK0059T ATCC 21756T, IF0 13240T IF0 Takahashi and Kawahara‘ Isolate K-207 TKO065 This study Isolate K-208 TKO066 This study 0. turbata TK0060T ATCC 25835T, DSM 20577T, IF0 13506T, JCM 3160T, NCIB JCM 105UT 0. xanthineolytica TK0061T ATCC 27402T, JCM 3WT JCM L. denitrificans TK0062T ATCC 14870T ATCC “A. luteus” TKO063 ATCC 21606 ATCC Kitamura et al.d A. globiformis ~~0064~ATCC 8010T, DSM 20124T, IAM 12438T, IF0 12137T, JCM 1332T, JCM NCIB 8907T a ATCC, American Type Culture Collection, Rockville, Md. ; DSM, Deutsche Sammlung von Mikroorganismen, Gottingen, Federal Republic of Germany; IAM, Institute of Applied Microbiology, University of Tokyo, Tokyo, Japan; IFO, Institute for Fermentation, Osaka, Japan; JCM, Japan Collection of Microorganisms, The Institute of Physical and Chemical Research, Wako-shi, Saitama, Japan; NCIB, National Collection of Industrial Bacteria, Tony Research Station, Aberdeen, United Kingdom. T. Takahashi, Y. Yamazaki, K. Kato, and M. Isono, U.S. patent 3,816,253, June 1974. T. Takahashi and K. Kawahara, U.S. patent 3,749,641, July 1973. K. Kitamura, T. Kaneko, Y. Yamamoto, and Y. Kuroiwa, U.S. patent 3,716,452, February 1973. FIG. 1. Negatively stained cells showing peritrichous flagella. (A) M. dimorpha TKOOST. (B) M. bullata TK0051T. Bars = 1 prn. 436 URAKAMI ET AL. INT. J. SYST.BACTERIOL. FIG. 2. Negatively stained cells exhibiting branching. (A) M. dimorpha TK0055T. (B) M. bullata TKO051T. Bars = 1 Fm. assay Casamino Acids; Difco Laboratories, Detroit, Mich.). TK0051T, TK0053T, TKOOST, TK0058, TK005gT, TK0065, Utilization of carbon compounds was determined in liquid and TKO066 basal medium B with the methanol replaced by other com- pounds, as described previously (18). The utilization of L-glutamic acid, L-asparatic acid, acetic acid, citric acid, RESULTS succinic acid, formic acid, n-butanol, putrescine, spermi- dine, and spermine was tested in the same manner, except Phenotypic characteristics of Mycoplana strains and iso- that the concentrations of the carbon compounds were 0.4% lates. All of the strains were gram-negative, nonsporeform- (wt/vol). Utilization of butane was tested in an atmosphere ing, rod-shaped organisms (0.5 to 0.8 by 2.0 to 3.0 pm) with containing C4HI0, 0,, and C02 (5:4:1) by using a rotary rounded ends. The cells occurred singly or rarely in pairs shaker and a tightly stoppered 1,000-ml conical flask con- and were motile by means of peritrichous flagella. The taining 300 ml of medium B. All bacteria were also grown in peritrichous flagella of M. dimorpha TKOOST and M. buiiata PYG broth containing 0.5% (wthol) peptone, 0.5% (wthol) TK0051T are shown in Fig. 1. Furthermore, these bacteria yeast extract, and 0.5% (wthol) glucose (pH 7.0) at 30°C for formed branching filaments prior to fragmentation (Fig. 2). 1 day with shaking, and the cells grown under these condi- Colonies were white to light yellow. Abundant growth tions were used to determine cellular fatty acid composi- occurred in nutrient broth, PYG broth, and peptone water. A tions, quinone systems, DNA base compositions, and levels water-soluble fluorescent pigment was not produced on King of DNA-DNA homology. A and King B media. Granules of poly-P-hydroxybutyrate Cellular fatty acid compositions. Cellular fatty acid com- accumulated in the cells. Production of indole in 1%tryptone positions were determined as described previously (19). broth and in the presence of hydrogen sulfide (TSI medium) Quinone systems and quinone homologs. Respiratory qui- was not observed. Hydrolysis of gelatin and starch was not nones were determined as described previously (17). observed. Ammonia was produced in peptone water. Deni- DNA base compositions. DNAs were extracted by using trification on PYG agar containing 0.3% (wthol) KNO, was the method of Saito and Miura (12), and guanine-plus- negative. Litmus milk was not changed. All strains utilized cytosine (G + C) contents were determined by reverse-phase peptone and Casamino Acids as carbon sources and nitrogen high-performance liquid chromatography (15).
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