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 Mycoplana and Description of Two New Species, 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 . 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). sources. Urease was not produced. Growth did not occur in DNA-DNA hybridization. DNA-DNA hybridization was the presence of 3% (wthol) NaC1. All strains produced acid carried out at 68°C by using the method of Kaneko et al. (8). from carbohydrates oxidatively , but not fermentatively. The DNAs from strains TKOOST, TK0053T, TK0051T, and biochemical and physiological characteristics that differed in TK005gT were labeled with [1',2',5-3H]dCTP by the nick the Mycoplana strains are shown in Table 2. translation method, using a type TK700 kit (Amersham The Mycoplanu strains and isolates were divided into four International plc, Amersham, United Kingdom). DNA-DNA groups on the basis of mucosity of colonies, oxidative hybridization experiments were performed with strains production of acid from sugars, requirement for growth VOL.40, 1990 NEW MYCOPLANA SPECIES 437

factor, utilization of carbon compounds, and utilization of nitrogen sources (Tables 2 and 3). Group 1 consisted of M. dimorpha TK0055’ (= ATCC 4279T), TKOO56’ (= IF0 13291’), and TK0057T (= NCIB 9439T) and isolate TK0066. Group 2 consisted of M. bullata TK0053T (= NCIB 9440T). Group 3 consisted of M. bullata TK0051’ (= ATCC 4278’) and TK0052T (= IF0 13290’) and isolate TK0065. Group 4 consisted of Mycoplana sp. strain TKO059’ and M. dimorpha TK0058. Colonies of organisms Nitrate belonging to groups 1and 2 were not mucous, but colonies of reduction organisms belonging to groups 3 and 4 were. Production of acid from sugars differed among the groups (Table 3). The Methyl red group 1bacteria required biotin for growth, the single group 2 test strain required thiamine, the group 3 bacteria required calcium pantothenate, and the group 4 bacteria required riboflavin. Voges-Proskauer test The bacteria belonging to groups 1 and 2 grew in the presence of L-arabinose, D-xylose, D-glucose, D-mannose, ti.!+ ++*++++++ Oxidase D-fructose, D-galactose, D-sorbitol, D-mannitol, glycerol, ii: L-glutamic acid, L-asparatic acid, acetic acid, putrescine, + +I IE+++++ Catalase a spermidine, and spermine, but did not grow in the presence ,p: I I I I I I++++ Biotin of lactose, trehalose, inositol, soluble starch, citric acid, E SF formic acid, methanol, ethanol, butanol, monomethylamine, I II11+1III Thiamine [2 $* methane, and butane. The group 1 bacteria grew in the WE3 presence of maltose, sucrose, and succinic acid, but the Calcium L I 1+++1IIII pantothenate P group 2 bacterium did not (Table 2). cd The bacteria belonging to groups 3 and 4 did not grow in Riboflavin medium containing 0.2 g of Casamino Acids per liter and the 9o_ compounds tested as carbon sources. These bacteria may salt ?2. require unknown growth factors which were not present in c the vitamins and amino acids which we tested. Thus, utili- Nitrate 05 zation of these carbon compounds, as well as utilization of li ammonium salts, nitrate, and urea as nitrogen sources, could Urea 2 not be determined. 8 Cellular fatty acid compositions. The cellular fatty acids of af t;‘ Peptone c. Mycoplana strains and the other isolates consisted of large amounts of n-C18:, acid, as well as n-C16:, and n-C,,:, acids Casamino (Table 4). + +++++++++ Acids x . In contrast, the cellular fatty acids of Oerskovia turbata e TK0060T, Oerskovia xanthineolytica TK006lT, Listeria den- Maltose S itrificans TK0062T, “Arthrobacter luteus” TK0063, and Ar- 0 throbacterglobifomis TK0064’ consisted of large amounts of v1 CT Sucrose anteiso-C,,:, acid and small amounts of iso-ClS:,, anteiso- &. C17:,, lt-C14:1, IZ-C~~,~,and cyclopropane-C,,,, acids (Table 5). v) Succinic acid Hydroxy fatty acid compositions. Bacteria belonging to groups 1 and 2 contained large amounts of 3-hydroxy-C,,,, acid. In contrast, bacteria belonging to groups 3 and 4 contained large amounts of 3-hydroxy-C,,,, acid (Tables 4 and 6). Quinone systems and quinone homologs. The Mycoplana + +++++++++ strains contained large amounts of ubiquinone Q-10 and small amounts of ubiquinones Q-9 and Q-11 (Table 7). In + +++++++++ contrast, 0. turbata, 0. xanthineolytica , L. denitrificans, “A. luteus,” and A. globifomis contained large amounts of + +++++++++ menaquinone MK-9 and small amounts of menaquinones 9 MK-8 and MK-10. However, these five bacteria could be 1 ll**IE1*8 divided into three types on the basis of degree of hydroge- ‘8cc 1 nation. Menaquinone MK-9 was found in the Listeria strain, I IIIIIIIII P, menaquinone MK-9(H2) was found in the Arthrobacter strain, and menaquinone MK-9(H4) was found in the Oer- + +++++++++ sbvia strains (Table 7). DNA base compositions. The DNA base compositions of + ++II++l++ Mycoplana strains and isolates ranged from 63 to 68 mol% G+C (Table 6). In contrast, the DNA base compositions of the Oerskovia strains ranged from 72 to 75 mol% G+C, and I IIIIIIIII the DNA base compositions of L. denitrificans TK0062T, 438 URAKAMI ET AL. Im. J. SYST.BACTERIOL.

TABLE 3. Oxidative production of acid from sugars by Mycoplana strains Acid produced from:

M. dimorpha TK0055T +u W + + + + M. dimorpha TK0056T + + + + + + M. dimorpha TK0057T + + + + + + TKO066 + + + + + + M. bullata TK0053T + + + + + + M. bullata TK0051T - - + - - - M. bullata TK0052T - - + - - - TKO065 - - + - - - Mycoplana sp. strain + + + + + + TK0059T M. dimorpha TKO058 + + + + + +

a +, Positive; -, negative; w, weak.

“A. luteus” TK0063, and A. globifomis TK0064T were 56, TK0056T (= IF0 13291T),and TK0057= (= NCIB 9439T) and 74.3, and 62.0 mol% G+C, respectively (Table 6). isolate TK0066. Group 2 consisted of M. bullata TK0053T (= DNA-DNA hybridization. The levels of DNA-DNA homol- NCIB 9440T). Group 3 contained M. bullata TK0051T (= ogy among Mycoplana strains and isolates are shown in ATCC 4278T) and TK0052T (= IF0 13290T) and isolate Table 8. The bacteria were clearly divided into four groups. TK0065. Group 4 contained Mycoplana sp. strain TK005gT These groups were identical to the groups based on physio- and M. dimorpha TK0058. logical characteristics (Tables 2 and 3), hydroxy fatty acid We believe that the Mycoplana strains which we studied compositions (Tables 4 and 6), and DNA base compositions can be separated into four distinct species that correlate with (Table 6). groups 1 to 4. Group 1 contains strains ATCC 4279T, IF0 13291T, and NCIB 9439T of M. dimorpha. Group 2 contains DISCUSSION strain NCIB 9440T of M. bullata, and group 3 contains The Mycoplana strains (6) and isolates TKO065 and strains ATCC 427gT and IF0 13290T of M. bullata. Although TKO066 shared the same morphological characteristics. Fur- strains NCIB 9440T, ATCC 427ST, and IF0 13290T of M. thermore, these bacteria shared the same major components bullata supposedly originated from the same source (6), of their cellular fatty acids and ubiquinone systems (Tables 4 these bacteria could be divided into the following two and 7). On the other hand, these organisms were divided into groups: group 2 (strain NCIB 9440T) and group 3 (strains four groups on the basis of mucosity of colonies, oxidative ATCC 4278T and IF0 13290T). The reason for this unex- production of acid from sugars, utilization of carbon com- pected observation is not apparent. pounds, and utilization of nitrogen sources (Tables 2 and 3), P. Pienta of the American Type Culture Collection and this division correlated well with DNA base composi- (ATCC) (personal communication) commented on the his- tions (Table 6), hydroxy fatty acid compositions (Tables 4 tory of one strain as follows: “Strain ATCC 4278 was and 6), and levels of DNA-DNA homology (Table 8). Group deposited by H. J. Conn of the New York Agricultural 1 contained M. dimorpha TK005ST (= ATCC 4279T), Experiment Station on April 4, 1928. The documentations

TABLE 4. Cellular fatty acid compositions of Mycoplana strains

Fatty acid composition (% of total acids)

Straight-chain acids Cyclopropane Strain acids 3-Hydroxy acids 3-OH- 3-OH- c14:0 c15:O c160 C16:l c17:0 c180 c18:1 c19:0 c17:0 c19:0 c12:o cldC0

M. dimorpha TK0055T 1.1 4.5 13.7 1.0 76.5 1.2 2.0 M. dimorpha TK0056T 5.6 22.0 0.3 0.5 69.8 0.4 1.1 M. dimorpha TK0057T 1.2 1.2 4.7 3.0 3.3 2.6 78.6 3.0 2.4 TKO066 5.7 24.2 0.2 0.4 67.9 0.4 0.1 1.1 M. bullata TK0053T 0.1 1.2 9.3 1.8 3.3 7.6 71.3 0.6 2.3 2.5 M. bullata TK005IT 2.6 2.4 20.3 6.0 2.8 0.3 62.9 0.3 2.4 M. bullata TK0052T 2.9 0.5 20.0 9.0 0.7 64.8 2.1 TKO065 24.3 5.5 1.2 0.6 66.0 0.5 0.1 2.0 Mycoplana sp. strain 0.2 19.8 17.8 0.2 0.3 57.2 ’ 0.5 1.8 2.0 TK0059T M. dimorpha TKO058 0.1 0.2 20.9 18.0 0.2 0.2 56.3 0.2 0.8 1.3 1.8 VOL. 40, 1990 NEW MYCOPLANA SPECIES 439

TABLE 5. Cellular fatty acid compositions of Listeria, Arthrobacter, and Oerskovia strains

Fatty acid composition (% of total acids) Is0 acids Anteiso acids Straight-chain acids Strain C15:O Iso- Iso- Anteiso- Anteiso- Anteiso- cy clopropane c14:0 c14:1 c15:0 c16:0 acid clS:O c17:0 c13:0 C15:O C1,:O

~ ~ ~~~~ L. denitrificans TK0062T 3 .O 2.8 58.2 2.0 4.2 7.6 14.7 7.5 A. globiformis TK0064T 3.4 0.8 52.8 18.0 1.7 1.1 6.6 15.6 “A. luteus” TKO063 6.5 0.3 55.1 15.9 0.6 3.0 10.6 8.0 0. turbata TK0060T 1 .o 41.1 16.9 2.6 4.5 7.7 11.0 15.2 0. xanthineolytica TK006lT 4.7 0.4 56.8 16.3 0.5 4.7 12.0 4.4 sent with the strain at that time indicated that he had ing rod), as shown in Fig. 1, and this strain is included in the obtained the strain from P. Gray shortly before that date. genus Mycoplana. Furthermore, strain ATCC 4278 is men- The strain carried the designation of ‘79’. ATCC 4278 was tioned as a type strain of M. bullata on the Approved Lists deposited in the IF0 as IF0 13290.” On the other hand, of Bacterial Names (13). Considering the information from P. N. Green of National Collection of Industrial and Marine Pienta of the ATCC and Green of the NCIB, strain ATCC Bacteria (NCIB) (personal communication) wrote as fol- 4278* seems to have clearer origin than strain NCIB 9440T. lows: “NCIB 9440 (strain M51) was deposited in the collec- Therefore, we believed that strain ATCC 4278T (= TK0051T) tion in 1963, having been received for research purposes in should be retained as the type strain of M. bullata. On the 1956 from P. H. H. Gray, Macdonald College, Quebec, basis of the data presented here, it seems reasonable to Canada. Gray originally isolated this strain when he worked conclude that the group 1 bacteria should be designated M. at Rothamsted Experimental Station, England. M51 is a dimorpha, that the group 3 bacteria should be designated M. Macdonald College number and not Gray’s original strain bullata, and that groups 2 and 4 are new species. designation. NCIB 9439 and M. bul- Mycoplana strains are clearly separated from the genera lata NCIB 9440, as far as the records show, are claimed to be Oerskovia, Listeria, and Arthrobacter (Tables 5 to 7). On the equivalent to ATCC 4279 and ATCC 4278, respectively. other hand, Mycoplana strains resemble Methylobacterium While I can confirm from my studies that the two M. and Xunthobacter strains on the basis of formation of dimorpha strains do appear identical phenotypically, the M. branching filaments (1, 7, 16, 21) and chemosystematic data bullata strains are quite different. In addition, we find that (16, 17, 19, 20). Therefore, these genera should be included while the NCIB strain is morphologically a typical Myco- in some higher taxon. However, Mycoplana strains are plana (branching rod), the ATCC strain is quite different and distinguished from Methylobacterium and Xanthobacter does not appear to branch. In our opinion ATCC 4278 is not strains by point of flagellation, colony color, utilization of a Mycoplana. This may be explained by the fact that Gray methanol, and hydroxy fatty acid composition (Table 9). and Thornton originally isolated two strains of M. bullata Since only limited information concerning the character- and it is possible NCIB and ATCC acquired different strains. istics of Mycoplana strains has been published previously (4, Therefore, it would be our opinion that NCIB 9440, and not 12,16), we present below emended descriptions of the genus ATCC 4278, should be considered as the reference strain Mycoplana and of M. dimorpha and M. bullata and propose from this species.” two new species in the genus, M. ramosa and M. segnis. Thus, there is a question whether strains ATCC 4278T and Emended description of Mycoplanu Gray and Thornton NCIB 9440* have the same origin. However, strain ATCC 1928. Cells are gram-negative and nonsporeforming, and 4278= is morphologically a typical Mycoplana strain (branch- branching filaments fragment into irregular rods (0.5 to 0.8

TABLE 6. DNA base compositions and hydroxy fatty acid compositions of Mycoplana, Listeria, Arthrobacter, and Oerskovia strains 3-Hydroxy acid composition G+C Major (% of total 3-hydroxy fatty acids) Strain content fatty (rnol%) acid 3-OH- 3-OH- 3-OH- 3-OH- 3-OH- C12:O c13:0 c14:0 C14:l C16:O

~ ~~

M. dimorpha TK0055= 64.5 ‘18:l 100 M. dimorpha TK0056T 64.4 C18:l 94.9 5.1 M. dimorpha TK0057T 64.4 c18:1 7.9 84.9 9.2 TKO066 64.4 Cl,:, 100 M. bullata TK0053T 63.7 c18:1 100 M. hdlata TK0051T 66.7 C18:l 92.4 4.0 3.6 M. bullata TK0052T 67.0 c18:1 94.5 5.5 TKO065 67.7 C18:l 92.9 7.1 Mycoplana sp. strain TK0059T 67.5 28.4 M. dimorpha TKO058 67.4 14.4 L. denitrificans TK0062= 56.0 A. globiformis TK0064T 62.0 “A. luteus” TKO063 74.3 Anteiso-C,,:, 0. turbata TK0060T 74.2 Anteiso-C,,:, 0.xanthineolytica TK0061T 72.3 Anteiso-C,,,, 440 URAKAMI ET AL. INT. J. SYST. BACTERIOL.

TABLE 7. Quinone types of Mycoplana, Listeria, Arthrobacter, and Oerskovia strains Quinone composition (% of total quinones) Quinone Ubiquinones” Menaquinonesb Strain Q-9 Q-lo Q-ll MK-7 MK-7 MK-8 MK-8 MK-8 MK-9 MK-9 MK-9 MK-10 MK-10 (Ho) (HJ (Hd (Hd (H4) (Ho) (Hd (H4) (Ho) (H2) M. dimorpha TKOOST Q-10 5.0 94.6 0.4 M. dimorpha TK0056T Q-10 2.7 96.9 0.4 M. dimorpha TK0057T Q-10 4.9 94.8 0.3 TKO066 Q-10 1.5 98.2 0.3 M. bullata TK0053T Q-10 5.3 94.4 0.3 M. bullata TKO051T Q-10 3.2 95.1 1.7 M. bullata TK0052T Q-10 0.8 97.7 1.5 TKO065 Q-10 1.2 98.0 0.8 Mycoplana sp. strain Q-10 8.4 89.2 2.4 TK0059T M. dimorpha TKO058 Q-10 8.6 90.3 1.1 L. denitrificans TK0062T MK-9 4.6 95.2 0.2 A. globiformis TK0064T MK-9(H,) 0.3 3.2 0.5 24.1 0.8 70.6 0.4 “A. luteus” TKO063 MK-9(H,) 1.5 1.2 86.3 11.0 0. turbata TK0060T MK-B(H4) 3.9 10.0 3.7 8.8 0.9 72.7 0. xanthineolytica TK0061T MK-9(H4) 0.1 3.4 5.8 4.4 0.2 85.0 1.1 The designations used for ubiquinones indicate the number of isoprene units in a side chain (e.g., ubiquinone Q-9 has nine isoprene units in a side chain). The designations used for menaquinones indicate the number of isoprene units in a side chain (e.g., menaquinone MK-8 has eight isoprene units in a side chain). Ho, H2, and H4 indicate the degree of hydrogenation. pm wide by 2.0 to 3.0 Fm long) that bear peritrichous Descriptions of M. dimorpha, M. ramosa, M. bullata, and flagella. Colonies are white to light yellow. Cells grow in M. segnis are given below. nutrient broth, PYG broth, and peptone water. A water- Emended description of Mycoplana dimorpha Gray and soluble fluorescent pigment is not produced on King A and Thornton 1928. Reduction of nitrate is variable. The Voges- King B media. Granules of poly-P-hydroxybutyrate accumu- Proskauer test is positive. Catalase positive. Acid is pro- late in the cells. The methyl red test is negative. Indole and duced from L-arabinose, D-xylose, D-glucose, D-mannose, hydrogen sulfide are not produced. Hydrolysis of gelatin and D-fructose, D-galactose, D-sorbitol, D-mannitol, and glycerol starch does not occur. Ammonia is produced. Denitrification oxidatively, but acid is not produced from maltose, sucrose, is negative. Litmus milk is not changed. Acid is produced lactose, trehalose, inositol, and soluble starch. L-Arabinose, from sugars oxidatively, but not fermentatively . Peptone and D-xylose, D-glucose, D-mannose, D-fructose, D-galactose, Casamino Acids are utilized as carbon sources and nitrogen maltose, sucrose, D-sorbitol, D-mannitol, glycerol, succinic sources. Urease and oxidase are produced. Aerobic. Metab- acid, L-glutamic acid, L-asparatic acid, acetic acid, pu- olism is strictly respiratory and not fermentative. Good trescine, spermidine, and spermine are utilized as sole growth occurs between pH 6.0 and 8.0 and at 30°C. Growth sources of carbon, but lactose, trehalose, inositol, soluble does not occur in medium containing 3% sodium chloride. starch, citric acid, formic acid, methanol, ethanol, n-buta- The DNA base composition ranges from 63 to 68 mol% nol, monomethylamine, methane, and butane are not. Biotin G+C. The cellular fatty acids consist of large amounts of is required for growth. Ammonia and urea are utilized as sole octadecenoic (CI8:,) acid, as well as hexadecanoic (c16:O) sources of nitrogen. Most strains utilize nitrate as a sole and hexadecenoic (&:I) acids. The major ubiquinone is nitrogen source. The DNA base composition ranges from 64 ubiquinone Q-lo. to 65 mol% G+C. The major hydroxy fatty acid is 3-hy- The type species is M. dimorpha Gray and Thornton 1928. droxy-C,,,, acid. The differential characteristics of Mycoplana species and The type strain is strain TKO055 (= ATCC 4279 = IF0 other genera of gram-negative branching bacteria are shown in Table 9. 13291 = NCIB 9439), which was isolated from soil by Gray and Thornton in 1928. The DNA base composition of the type strain is 64.5 mol% G+C. Isolate TKO066 is also an M. TABLE 8. Levels of DNA-DNA homology among dimurpha strain. Mycoplana strains Emended description of Mycoplana buZZata Gray and Thorn- ton 1928. Reduction of nitrate is weak or negative. The % DNA-DNA homology with: Voges-Proskauer test is positive. Catalase activity is weak or Strain Strain Strain Strain Strain negative. Acid is produced from D-glucose oxidatively, but TKO05ST TK0053T TK0051T TK0059T is not produced from D-arabinose, D-xylose, D-mannose, D- M. dirnorpha TKOOST 100 39 9 11 fructose, D-galactose, maltose, sucrose, lactose, trehalose, TKO066 102 42 10 12 D-sorbitol, D-mannitol, inositol, glycerol, and soluble starch. M. bullata TK0053* 47 100 9 11 Calcium pantothenate is required for growth. The utilization M. bullata TK0051T 22 14 100 29 of single carbon compounds and nitrogen compounds cannot TKO065 20 11 61 34 be determined at this time, possibly because of axotrophy. Mycoplana sp. strain 13 6 15 100 The DNA base composition ranges from 66 to 68 mol% TK0059T M. dimorpha TKO058 12 6 14 92 G+C. The major hydroxy fatty acid is 3-hydroxy-C,,,, acid. The type strain is strain TKO051 (= ATCC 4278 = IF0 VOL. 40, 1990 NEW MYCOPLANA SPECIES 441

TABLE 9. Characteristics which differentiate Mycoplana species from related genera

~ ~~~ ~ Color Mucosity Utilization of Major G+C Genus Species Motility of of hydroxy content colonies colonies Methanol Hydrogen acid (mol%)

~~ Mycoplana M. dimorpha + (Peritrichous) White - - - 3-oH-C,4,0 64-65 M. ramosa + (Peritrichous) White - - - 3-oH-C14:0 63.7 M. bullata + (Peritrichous) White + - - 3-OH-C12,0 67-68 M. segnis + (Peritrichous) White + - - 3-OH-C12:, 67-68 Methylobacterium M. extorquens + (Polar) Pink - + - 3-oH-C14:0 65-67 Xanthobacter X. autotrophicus - Yellow - + + 3-oH-C1,,0 6-9

13290), which was isolated from soil by Gray and Thornton 2. Breed, R. S., R. G. E. Murray, and P. H. Smith (ed.). 1957. in 1928. The DNA base composition of the type strain is 66.7 Bergey’s manual of determinative bacteriology, 7th ed. The mol% G+C. Isolate TKO065 is also an M. bullata strain. Williams & Wilkins Co., Baltimore. Description of Mycoplana ramosa sp. nov. My coplana 3. Buchanan, R. E., and N. E. Gibbons (ed.). 1974. Bergey’s manual of determinative bacteriology, 8th ed. The Williams & ramosa (ra.mo’sa. M.L. ramosa, ramous, ramose, Wilkins Co., Baltimore. branched, branchlike). Positive for reduction of nitrate, the 4. Collins, M. D. C., and M. Goodfellow. 1979. Isoprenoid qui- Voges-Proskauer test, and catalase activity. Acid is pro- nones in the classification of coryneform and related bacteria. J. duced from L-arabinose, D-xylose, D-glucose, D-mannose, Gen. Microbiol. 110:127-136. D-fructose, D-galactose, D-sorbitol, D-mannitol, and glycerol 5. Cross, T., and M. Goodfellow. 1973. and classifica- oxidatively, but is not produced from maltose, sucrose, tion of the actinomycetes, p. 11-112. In G. Sykes and F. A. lactose, trehalose, inositol, and soluble starch. L-Arabinose, Skinner (ed.), Actinomycetales: characteristics and practical D-xylose, D-glucose, D-mannose, D-fructose, D-galactose, importance. Academic Press, Inc., New York. D-sorbitol, D-mannitol, glycerol, L-glutamic acid, L-asparatic 6. Gray, P. H. H., and H. G. Thornton. 1928. Soil bacteria decompose certain aromatic compounds. Zentralbl. Bakteriol. acid, acetic acid, putrescine, spermidine, and spermine are Parasitenkd. Infektionskr. Hyg. Abt. 2 73:74-96. utilized as sole sources of carbon, but maltose, sucrose, 7. Green, P. N., and I. J. Bousfield. 1982. A taxonomic study of lactose, trehalose, inositol, soluble starch, succinic acid, some gram-negative facultatively methylotrophic bacteria. J. citric acid, formic acid, methanol, ethanol, n-butanol, Gen. Microbiol. 128:623438. monomethylamine, methane, and butane are not. The DNA 8. Kaneko, T., R. Nozaki, and K. Aizawa. 1978. Deoxyribonucleic base composition is 64 mol% G+C. The major hydroxy fatty acid relatedness between Bacillus anthracis, Bacillus cereus, acid is 3-hydroxy-C,,,, acid. and Bacillus thuringiensis. Microbiol. Immunol. 22:639441. The type strain is strain TKO053 (= NCIB 9440), which is 9. Lechevalier, H. A., and M. P. Lechevalier. 1981. Introduction to strain M51 of Macdonald College and was isolated from soil the order Actinomycetales, p, 1915-1922. In M. P. Starr, H. Stolp, H. G. Triiper, A. Balows, and H. G. Schlegel (ed.), The by Gray and Thornton in 1928. This strain was deposited prokaryotes, a handbook on habitats, isolation, and identifica- previously in the NCIB as an M. bullata strain. tion of bacteria, vol. 2. Springer-Verlag, AG, Berlin. Description of Mycoplana segnis sp. nov. Mycoplana segnis 10. Lechevalier, H. A,, and M. P. Lechevalier. 1981. Actinomycete (seg‘nis. L.adj. segnis, slow, sluggish, inactive). Nitrate is genera “in search of a family,” p. 2118-2119. In M. P. Starr, H. not reduced to nitrite. The Voges-Proskauer test is negative Stolp, H. G. Triiper, A. Balows, and H. G. Schlegel (ed.), The (strain TK0059T does not grow in glucose phosphate broth). prokaryotes, a handbook on habitats, isolation, and identifica- Catalase positive. Acid is produced from L-arabinose, D-XY- tion of bacteria, vol. 2. Springer-Verlag, AG, Berlin. lose, D-glucose, D-mannose, D-fructose, D-galactose, malt- 11. Lechevalier, H. A., and M. P. Lechevalier. 1987. Genus Oer- ose, sucrose, lactose, and trehalose oxidatively, but is not skovia Prauser, Lechevalier and Lechevalier 1970,534; emended Lechevalier 1972,263AL, p. 1489-1491. In P. H. A. produced from D-sorbitol, D-mannitol, inositol, glycerol, and Sneath, N. S. Mair, M. E. Sharpe, and J. G. Holt (ed.), Bergey’s soluble starch. Riboflavin is required for growth. The utili- manual of systematic bacteriology, vol. 2. The Williams & zation of single carbon compounds and nitrogen compounds Wilkins Co., Baltimore. cannot be determined at this time, possibly because of 12. Saito, H., and K. Miura. 1963. Preparation of transforming axotrophy. The DNA base composition ranges from 67 to 68 deoxyribonucleic acid by phenol treatment. Biochim. Biophys. mol% G+C. The major hydroxy fatty acid is 3-hydroxy-C,,,, Acta 72:619-629. acid. 13. Skerman, V. B. D., V. McGowan, and P. H. A. Sneath (ed.). The type strain is strain TKO059 (= ATCC 21756 = IF0 1980. Approved lists of bacterial names. Int. J. Syst. Bacteriol. 13240), which was isolated from soil by Takahashi and 30:225-420. 14. Sukapure, R. S., M. P. Lechevalier, H. Reber, M. L. Higgins, Komahara in 1973. The DNA base composition of the type H. A. Lechevalier, and H. Prauser. 1970. Motile nocardioid strain is 67.5 mol% G+C. Strain TKO058 (= ATCC 21759 = Actinomycetales. Appl. Microbiol. 19527-533. IF0 13213),which was previously assigned to the species M. 15. Tamaoka, J., and K. Komagata. 1984. Determination of DNA dimorpha, is a strain of M. segnis. base composition by reversed-phase high-performance liquid chromatography. FEMS Microbiol. Lett. 25125-128. ACKNOWLEDGMENTS 16. Urakami, T., and K. Komagata. 1984. Protomonas, a new genus We thank P. Pienta (ATCC) and P. N. Green (NCIB) for assis- of facultatively methylotrophic bacteria. Int. J. Syst. Bacteriol. tance concerning the history of the type strain of M. bullata. W.188-201. 17. Urakami, T., and K. Komagata. 1986. Occurrence of isoprenoid LITERATURE CITED compounds in gram-negative methanol-, methane-, and methyl- 1. Bousfield, I. J., and P. N. Green. 1985. Reclassification of bacteria amine-utilizing bacteria. J. Gen. Appl. Microbiol. 32:317-341. of the genus Protomonas Urakami and Komagata 1984 in the 18. Urakami, T., and K. Komagata. 1986. Methanol-utilizing Ancy- genus Methylobacterium (Patt, Cole, and Hanson) emend. Green lobacter strains and comparison of their cellular fatty acid and Bousfield 1983. Int. J. Syst. Bacteriol. 35209. compositions and quinone systems with those of Spirosorna, 442 URAKAMI ET AL. INT. J. SYST.BACTERIOL.

Flectobacillus, and Runella species. Int. J. Syst. Bacteriol. composition and DNA-DNA homologies of methanol-utilizing 36:415421. bacteria. J. Gen. Appl. Microbiol. 31:243-253. 19. Urakami, T., and K. Komagata. 1987. Cellular fatty acid com- 21. Wiegel, J. K., and H. G. Schlegel. 1984. Genus Xanthobacter position with special reference to the existence of hydroxy fatty Wiegel, Wilke, Baungarten, Opitz and Schlegel 1978,573*=, p. acids in gram-negative methanol-, methane-, and methylamine- 325-333. In N. R. Krieg and J. G. Holt (ed.), Bergey’s manual utilizing bacteria. J. Gen. Appl. Microbiol. 33:135-165. of systematic bacteriology, vol. 1. The Williams & Wilkins Co., 20. Urakami, T., J. Tamaoka, and K. Komagata. 1985. DNA base Baltimore.