The Genus Arthrobacter Was Established for Soil Coryneform Bacteria with a Life Cycle Different from Corynebacterium and Mycobacterium by Connand DIM- Mickin 1947 (1)

J. Gen. App!. Microbiol., 29, 59-71 (1983)

PIMELOBACTER GEN. NOV., A NEW GENUS OF CORYNEFORM BACTERIA WITH LL-DIAMINOPIMELIC ACID IN THE CELL WALL

KEN-ICHIRO SUZUKII AND KAZUO KOMAGATA

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

(Received December 22, 1982)

Coryneform bacteria with LL-diaminopimelic acid in the cell wall were characterized. Morphological, biochemical, and chemotaxonomic characteristics, and DNA homologies revealed that they form an independent cluster among the coryneform bacteria. A new genus Pimelobacter is proposed for this group of coryneform bacteria. The type species of the genus Pimelobacter is Pimelobacter simplex comb. nov., which was previously named Art hrobacter simplex. Further, Pimelobacter jensenii sp. nov. and Pimelobacter tumescens comb. nov. are also described.

The genus Arthrobacter was established for soil coryneform bacteria with a life cycle different from Corynebacterium and Mycobacterium by CONNand DIM- MICKin 1947 (1). The chemotaxonomic heterogeneity of the genus Arthrobacter was first pointed out by CUMMINSand HARRIS(2). Some strains have lysine in the cell wall and others have LL-diaminopimelic acid (LL-DAP). Later, FIEDLER et a!. (3) and YAMADAand KoMAGATA(4) reported that a majority of the Arthro- bacter strains, including the type species Arthrobacter globiformis, have lysine and a few strains have LL-DAP in the cell wall. YAMADAand K®MAGATA(4) also reported that the DNA base composition of Arthrobacter strains with lysine in the cell wall ranges from 58 to 65 mol % of guanine plus cytosine (mol 0 G-+ C), while the DNA base composition of the coryneform bacteria with LL-DAP in the cell wall ranges from 70 to 72 mol % G+C; but the two groups of bacteria resemble each other with respect to their morphological and biochemical characteristics. The study of DNA homologies show that the coryneform bacteria with LL-DAP in the cell wall fall into one cluster and 1 Present address : Japan Collection of Microorganisms , Institute of Physical and Chemical Research, Wako-shi, Saitama 351, Japan. Reprint requests to: Dr. K. Suzuki, Japan Collection of Microorganisms, Institute of Physical and Chemical Research, Wako-shi, Saitama 351, Japan.

59 60 SUZUKI and KOMAGATA VOL. 29 are clearly distinguished from Arthrobacter strains with lysine in the cell wall (S). On the other hand, PRAVSER(6) established the genus Nocardioides for the no- cardioform bacteria with LL-DAP in the cell wall and suggested a close relationship of the strains of this genus to Arthrobacter simplex, one of the coryneform bacteria with LL-DAP in the cell wall. The taxonomic position of the coryneform bacteria with LL-DAP in the cell wall (the LL-DAP coryneform bacteria) needs clarification. KEDDIEand BOUSFIELD (7) also pointed out the necessity of establishing a new genus for the group of bacteria on the basis of much more information. The present study deals with the characterization of the LL-DAP coryneform bacteria and the establishment of a new genus, Pimelobacter gen. nov.

MATERIALS AND METHODS

Bacterial strains. We studied the strains listed in Table 1. Art hrobacter simplex CNF 035 and Arthrobacter tumescens CNF 067 are the type strains of those species (8). They were isolated by JENSEN(9). A. simplex CNF 091 was also isolated by GUNDERSENand JENSEN(10) as a herbicide-decomposing bacterium. Two strains of "Brevibacterium lipolyticum," CN F 036 and CNF 037, were isolated by IIzUKA and KOMAGATA(11) from soils of an oil field in Japan. In addition to the five strains of the LL-DAP coryneform bacteria, six strains of Arthrobacter with lysine in the cell wall, six strains of Nocardioides albus, and the type strain of Streptomyces albus were studied. Corynebacterium diphtheriae CNF 017 (=AJ 1414=ATCC 11913) was employed as a reference for cell wall analysis, and Micrococcus luteus CNF 040 (=IAM 1056=ATCC 4698) was used as a reference for determination of DNA base composition. Names in quotation marks are not on the Approved Lists of Bacterial Names, 1980 (8). Morphological characteristics. Cell morphology was observed in the cells grown on YM agar, which contained 0.5 % Bacto-peptone (Difco), 0.3 % yeast extract (Difco), 0.3 % malt extract (Difco), 1 % glucose, and 2 % agar; and on glucose-asparagine agar (GA agar), which contained 1 % glucose, 0.5 % asparagine, and 0.5 % K2HPO4, and 2 % agar. The latter medium was used for mycelial for- mation in the strains studied. Biochemical characteristics. Extracellular DNase was tested using DNase test agar (Difco). Urease and assimilation of organic acids were examined by the methods of YAMADAand KOMAGATA(12). Assimilation of carbohydrates was tested by the method of the International Streptomyces Project (13). Cells grown on GA agar were used for inocula, except for A. tumescens CNF 067 which was grown on YM agar. Since A, tumescens requires thiamine (14), vitamins were added to the basal medium in the three procedures, N, T, and 0 as shown in Table 2. The N medium contained 10 ppm thiamine HCI, 1 ppm biotin, 10 ppm ribo- flavin, 10 ppm nicotinic acid, 10 ppm pyridoxin, and 10 ppm p-aminobenzoic acid. 1983 Pimelnhacter gen. nov. b1

Table 1. Bacterial strains studied. 62 SUZUKI and KOMACATA VOL. 29

The T medium contained 10 ppm thiamine HC1, and the 0 medium contained no nutritional additives. Principal amino acids in the cell wall. DAP isomers in the cell wall were determined by the method of STANECKand RoBERTS(15). Isoprenoid quinones. Isoprenoid quinone was analyzed by high performance liquid chromatography. The apparatus and the analytical conditions were the same as those reported by TAMAOKAet al. (16). The abbreviations used for Iso- prenoid quinones in the present study are : M K, menaquinone; MK-n with n de- noting a specified number of isoprene units in the side chain; MK-n(Hm) with m indicating the number of hydrogen atoms saturating the Isoprenoid chain. Cellular fatty acid composition. Cellular fatty acid composition was determined by the method previously reported (17-19). DNA base composition and DNA homologies. DNA was extracted by the method of SAITOand MIURA(20). DNA base composition was calculated by the formula of MARMURand DOTY(21) from the melting temperature in SSC (SSC: 0.15 M NaCI and 15 mM trisodium citrate, pH 7.0). DNA of Micrococcus luteus CNF 040 (= JAM 1056, =ATCC 4698), which contains 72.0 mol % GEC, was employed as a reference for determining the DNA base composition and for the negative control of DNA hybridization. DNA homologies were carried out by the method previously reported (5,17). The hybridization temperature was 70°.

RESULTS Morphological characteristics Pleomorphism occurred in the five strains of the LL-DAP coryneform bacteria (Fig. 1). The cells were irregularly rod-shaped in the early stage of growth and coccoid in old cultures. After one day of cultivation, the cells of A. simplex CNF 035 were 1.0 ,ccmthick and 1.5 to 6.0µm long (most of them were 2.0 to 3.0 ,um long). Two strains of "Brevibacterium lipolyticum" CNF 036 and CNF 037 tended to be coccoid earlier than the other LL-DAP coryneform bacteria. A.

Fig. 1. Morphological changes of cells in Pimelobacter simplex CNF 091. a) 1-day culture on YM agar, b) 7-day culture on YM agar. The bar indicates 10 tam. 1983 Pimelobacter gen, nov. 63

Fig. 2. Comparison of cell morphologies in Pimelobacter simplex strains and a Nocardioides albus strain. a) P, simplex CNF 035, (b) P, simplex CNF 091, and c) N, albus CNF 146. Cells of each strain were those cultivated on YM agar at 30° for 2 days. The bar indicates 10 um. globiformis CNF 022 was also pleomorphic and the cells were 1.2 to 1.5 µm by 1.5 to 7.0 um in one-day cultures. A comparison of the cell morphology is shown in Fig. 2. Strains of N. albus had a regular filamentous form and its fragmented rods, and they were not coccoid even in the old culture. The cells were 0.5 to 0.6 µm in diameter. The motility of A, simplex CNF 035 and "Brevibacterium lipolyticum" CNF 036 and CNF 037 was observed by the hanging drop method. Flagellation of these strains was reported previously to be lateral (12,14) or peritrichous (10). A. tumescens CNF 067 and A. simplex CNF 091 were not motile. The five strains of the LL-DAP coryneform bacteria formed white, glossy, smooth, and entire colonies on YM agar. On the other hand, the colonies of N. albus were white and entire, but wrinkled. Some strains of N. albus formed aerial mycelia on GA agar.

Biochemical characteristics The results of the biochemical tests are shown in Table 2. No strains produced acid from glucose. All of the LL-DAP coryneform bacteria except A. simplex CNF 091 were positive for DNase and negative for urease. Acetic acid was assimilated by all the strains studied. Citric acid was not assimilated by the four strains of the LL-DAP coryneform bacteria and by five strains of N. albus. The strains of the LL-DAP coryneform bacteria did not assimilate uric acid, while five strains of Arthrobacter with lysine in the cell wall and six strains of N. albus did so. All the strains studied grew on glucose and sucrose. A. tumescens CNF 067 required thiamine for growth. Among the strains of the LL-DAP coryneform bacteria, A. tumescens CNF 067 grew at the expense of mannitol, and A. simplex CNF 091 did so at the expense of L-rhamnose.

Principal amino acids in the cell wall It was confirmed that the five strains of the LL-DAP coryneform bacteria and the type strains of N. albus (CNF 146) and S. albus (CNF 090) had LL-DAP in the 64 SUZUKT and KOMACATA VOL. 29

Table 2. Biochemical characteristics of the LL-

whole cell. DAP isomers were not detected in the cells of A, globiformis CNF 022, and meso-DAP was found in the cells of C. diphtheriae CNF 017.

Isoprenoid quinones and cellular fatty acid composition The Isoprenoid quinones and the cellular fatty acid composition of the LL-DAP coryneform bacteria are shown in Table 3. The major isoprenoid quinone of the LL-DAP coryneform bacteria was the menaquinone of MK-8(H4). N. albus CNF 146 had not only MK-8(H4) but also MK-8(H2) and MK-8. The cellular fatty acid composition of the LL-DAP coryneform bacteria was the complex type previously reported (19). The major components of the cellular fatty acids of A. simplex CNF 035 and "B. lipolyticum" CNF 036 and CNF 037 were C18:1,anteiso-C17:o, and iso-C16:o. 2-Hydroxy fatty acids of anteiso-C17:o and iso-C18:o were contained in the cells of these strains. A trace amount of 2-hydroxy fatty acid was detected in the cells of A. tumescens CNF 067. Cellular fatty acid composition of N. albus 1983 Pimelobacter gen, nov. 65

CNF 146 resembled that of the LL-DAP coryneform bacteria but did not contain 2-hydroxy fatty acids.

DNA base composition and DNA homologies The DNA base composition and DNA homologies of the LL-DAP coryneform bacteria are shown in Table 4. The DNA base composition of the LL-DAP coryneform bacteria ranged from 68.8 to 73.5 mol % G+ C, while that of N. albus CNF 146 was 68.6 mol % GEC. A. simplex CNF 035 and "Brevibacterium lipolyticum" CNF 036 and CNF 037 showed high homology values each other, but no strains showed high homology indices to either A. tumescens CNF 067 or N. albus CNF 146. 66 SUZUKI and KOMAGATA VOL. 29 1983 Pimelohacter gen. nov. 67

Table 4. Genetic relatedness among the LL-DAP coryneform bacteria and related organisms.

DISCUSSION YAMAHAand KoMAGATA(4) divided 112 strains of coryneform bacteria into seven groups on the basis of their principal amino acids in the cell wall, DNA base composition, the mode of cell division, and physiological and biochemical characteristics. The group 6 in their grouping consists of six strains, which possess LL- DAP in the cell wall. But, they still retain the naming of this group of coryneform bacteria to date. Among the strains of the group 6, A. atrocyaneus AJ 1429 (=ATCC 13752) and A. variabilis AJ 1434 (=ATCC 15753) were found to contain not LL-DAP in the cell wall but L-lysine and meso-DAP, respectively (3, 22, 23); and they were eliminated from the group 6. The present study revealed various differences among the three groups of bacteria, the LL-DAP coryneform bacteria, N, albus strains, and the strains of Art hrobacter containing lysine in the cell wall. The morphological, biochemical, and chemotaxonomic characteristics have led to the establishment of a new genus for the five strains of the LL-DAP coryneform bacteria. The strains of the genus Arthrobacter, represented by A. globiformis with lysine in the cell wall, showed pleomorphism similar to the LL-DAP coryneform bacteria, but they are quite different from the latter in regard to the principal amino acid in the cell wall (3, 4, 22, 23), isoprenoid quinones (24, 25), DNA base 68 SUZUKT and KOMAGATA VOL. 29 composition (4, 26), and DNA homology (5). The type strain of A. simplex (CNF 035) with LL-DAP in the cell wall was distantly related to the strains of Arthro- bacter in 16S ribonucleic acid cataloging (27). Propionibacterium, a genus of anaerobic coryneform bacteria, shares the same principal amino acids, LL-DAP and glycine, in the cell wall with the LL-DAP coryneform bacteria (22, 28), but all the strains of Propionibacterium produce acid from glucose (29). The major cellular fatty acids of the strains of Propionibacterium are iso- or anteiso-C15:o (30). The Streptomyces species also possess LL-DAP in the cell wall (22, 31). How- ever, they are quite different from the LL-DAP coryneform bacteria in cell morphology and cellular fatty acid composition (Table 3). The Streptomyces species have menaquinone of MK-9(H8) or MK-9(H6) (32). The N. albus strains were rather closely related to the LL-DAP coryneform bacteria in the chemotaxonomic characteristics presented in this study. The N. albus strains do not contain 2-hydroxy fatty acids in the cells, and do contain the minor components of menaquinones MK-8(H2) and MK-8, which were not detected in the cells of the LL-DAP coryneform bacteria. Furthermore, the range of G+C content of DNA of the LL-DAP coryneform bacteria was higher than that of N. albus, and DNA homology clearly showed distant relationship between the two. Cell morphology and colony appearance are different between the N, albus strains and the LL-DAP coryneform bacteria, as mentioned above. Based on the characteristics reported in the present study and in the references, a new genus Pimelobacter is proposed here. The name of Pimelobacter comes from the complex type of cellular fatty acid composition and the isolation of the strains from oily habitats. A. simplex CNF 035 is used for the transformation of steroid compounds (33-37). "Brevibacterium lipolyticum" CNF 036 and CNF 037 were isolated from an oil field and utilize hydrocarbons (11). For the members of the genus Pimelobacter, P. simplex comb. nov., P, jensenii sp. nov., and P. tumescens comb. nov. are proposed. P. simplex is the type species of the genus and includes the three strains, CNF 035, CNF 036, and CNF 037. The strain CNF 035, the previous type strain of Arthrobacter simplex, is proposed as the type strain of P. simplex. Two strains of "Brevibacterium lipolyticum," CNF 036 and CNF 037, are reidentified as P. simplex. A. simplex CNF 091 is revealed to be different from the type strain of A. simplex, CNF 035, and is proposed as the type strain of a new species, P. jensenii. A. tumescens CNF 067 is distinct from the other strains studied in its nutritional requirement, biochemical tests, cellular fatty acid composition, and DNA homologies. A, tumescens CNF 067 is the type strain of P, tumescens.

Description of Pimelobacter Suzuki and Komagata gen. nov. Pi. me. lo. bac'ter : Gr. n. pimele oil (-inhabiting); M. L. n. batter the masculine equivalent of the Gr. neut. n. bacterium a rod; M. L. masc. n. Pimelobacter oil- inhabiting rod. 1983 Pimelobactergen, nov. 69

Irregular rods, 0.6 to 1.2 by 1.0 to 7.0 µm, are formed in one-day culture on YM agar. Initially, branching occurs, but it is not dominant. Small coccoids occur dominantly in old cultures. Aerial mycelia are not formed. Motile or non-motile. In case of motile, peritrichous flagella are found. Gram positive. Occasionally gram variable in old cultures. Not acid-fast. Colonies are glossy, smooth, entire, and white or yellowish white on YM agar. DNase is positive. Gelatin and casein are hydrolysed. Acid is not produced from sugars. Glucose and sucrose are assimilated. Various kinds of organic acids are assimilated. Some species have nutritional requirements. Aerobic. DNA base composition ranges from 68.8 to 73.5 mol % G+C. LL-Di- aminopimelic acid and glycine are the principal amino acids in the cell wall. Cel- lular fatty acid composition is the complex type composed of branched and straight chain fatty acids containing both saturated and monounsaturated acids. 2- Hydroxy fatty acids occur. The isoprenoid quinone is MK-8(H4). The type species is P„ simplex (Jensen) Suzuki and Komagata comb. nov.

Description of Pimelobacter simplex (Jensen) Suzuki and Komagata comb. nov. Basonym : Corynebacterium simplex Jensen 1934. Synonym : Arthrobacter simplex (Jensen 1934) Lockhead 1957. Irregular rods, 1.0 to 1.2 by 1.5 to 6.0 ,um, are formed in one-day culture on YM agar. Branching occurs in the early growth phase. Small coccoid forms occur dominantly in the old culture. Aerial mycelia are not formed. Motile with peritrichous flagella. Gram positive. Occasionally gram variable in the old culture. Colonies are glossy, smooth, entire, and slightly yellowish white on YM agar. DNase is positive. Urease negative. Gelatin and casein are hydrolysed. Acid is not produced from sugars. Glucose and sucrose are assimilated. L- Rhamnose and n-mannitol are not assimilated. Various kinds of organic acids are assimilated, but hippurate and ureate are not. No nutritional requirement. Aerobic. The range of DNA base composition is 72.0 to 73.5 mol % G+C. LL-Di- aminopimelic acid and glycine are the principal amino acids in the cell wall. Cel- lular fatty acid composition is the complex type containing 2-hydroxy fatty acids. The isoprenoid quinone is MK-8(H4). The type strain is P. simplex CNF 035 (=AJ 1420, =ATCC 6946, =IAM 1660, =JCM 1363). Strains examined: CNF 035, CNF 036, CNF 037.

Description of Pimelobacter jensenii Suzuki and Komagata sp. nov. jen, se'ni. i. M. L. gen, n, jensenii of Jensen; named for H. L. Jensen, the Danish bacteriologist who contributed to the taxonomy of coryneform bacteria. 70 SUZUKI and KOMAGATA VOL. 29

Irregular rods, 0.6 to 0.8 by 3.0 to 7.0 µm are formed in one-day culture and 0.6 to 1.0 by 0.8 to 1.0 µm in seven-day culture on YM agar. Branching is not distinctive. Small coccoid forms are found dominantly in old culture. Aerial mycelia are not formed. Non-motile. Gram positive. Colonies are glossy, smooth, entire, and slightly yellowish white on YM agar. DNase activity is weak. Urease is positive. Acid is not produced from sugars. Glucose, sucrose, and L-rhamnose are assimilated. Acetate is assimilated, but succinate is not. No nutritional requirement. Aerobic. The DNA base composition is 68.8 % G+C. LL-Diaminopimelic acid and glycine are the principal amino acids in the cell wall. Cellular fatty acid composition is the complex type. Small amounts of 2-hydroxy fatty acids are found. The type strain is P. jensenii CNF 091 (=NCIB 9770, =IAM 12581, =JCM 1364).

Description of Pimelobacter tumescens (Jensen) Suzuki and Komagata comb. nov. Basonym : Corynebacterium tumescens Jensen 1934. Synonym : Arthrobacter tumescens (Jensen 1934) Conn and Dimmick 1947. Irregular rods, 1.0 to 1.2 by 1.5 to 6.0µm, are formed in one-day culture on YM agar. Branching occurs dominantly in the early stage of the growth. Small coccoid forms are dominant in the old culture. Aerial mycelium is not formed. Non-motile. Gram positive. Occasionally gram variable in the old culture. Colonies are glossy, entire, smooth, and slightly yellowish white on YM agar. DNase is positive. Urease is negative. Various kinds of organic acids are assimilated, but citrate and ureate are not. Glucose, sucrose, and D-mannitol are assimilated. L-Arabinose and L-rhamnose are not assimilated. Thiamine is required for growth. Aerobic. DNA base composition is 71.3 % G+C. LL-Diaminopimelic acid and glycine are the principal amino acids in the cell wall. Cellular fatty acid composition is the complex type, and trace amounts of 2-hydroxy fatty acids are found. The isoprenoid quinone is MK-8(H4). The type strain is P. tumescens CNF 067 (=ATCC 6947, =IAM 12345, =JCM 1365).

For kindly supplying cultures, the authors wish to thank Dr. H. Prauser, Akademische der Wissenschaften der DDR, Zentralinstitut fur Mikrobiologie and experimentelle Therapie, Jena, German Democratic Republic; Dr. I. Bousfield, National Collection of Industrial Bacteria, Torry Research Station, Aberdeen, Scotland; and Dr. A. Seino, Kaken Pharmaceutical Co., Ltd., Tokyo, Japan. We are indebted to Prof. H. Kuraishi and Mr. J. Tamaoka, Tokyo University of Agri- culture and Technology, Tokyo, for analysis of isoprenoid quinones. The present study was supported by the grants from the Waksman Foundation of Japan, Inc., Tokyo.

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