Morphologically Distinct Actinomycetes

Transcription

1 JOuRNAL OF BACTERIOLOGY, Feb., 1965 Copyright 1965 American Society for Microbiology Vol. 89, No. 2 Printed in U.S.A. Comparison of the Cell-Wall Composition of Morphologically Distinct Actinomycetes TATSURO YAMAGUCHI Institute of Applied Microbiology, The University of Tokyo, Bunkyo-ku, Tokyo, Japan Received for publication 1 October 1964 ABSTRACT YAMAGUCHI, TATSURO (The University of Tokyo, Tokyo, Japan). Comparison of the cell-wall composition of morphologically distinct actinomycetes. J. Bacteriol. 89: Cell-wall composition of various morphologically distinct actinomycetes was studied to determine the relationship, if any, between cell-wall composition and morphological criteria in actinomycete taxonomy. The methods used were similar to those of Cummins and Harris. At least five types of cell-wall composition were obtained; however, these were not always correlated with groupings by the conventional classification system. For instance, the sporangium-forming actinomycetes, Actinoplanaceae, had three types of cell-wall composition; the composition of cell walls of Promicromonospora, Micromonospora, and Microbispora was the same as, or similar to, that of Actinomyces, Actinoplanes, and Streptosporangium, respectively; Chainia, Actinopycnidium, Actinosporangium, and Microellobosporia had the same cell-wall composition as Streptomyces, whereas that of Streptoverticillium was slightly different. Possible implications of cell-wall composition and morphological differentiation of hyphae for the taxonomy and phylogeny of actinomycetes are also discussed. Actinomycetes are morphologically well-differentiated organisms, ranging from the simple, rapidly fragmenting, soft Nocardia to more complex forms having aerial hyphae, sclerotic granules, sporangia, or pycnidium-like fruiting bodies. Recent reports on Amorphosporangium, Ampullariella, and Spirillospora (Couch, 1963, 1964), Promicromonospora (Krassilnikov, Kalakoutskii, and Kirillova, 1961), Actinosporangium (Krassilnikov and Tsi-Shen, 1961), Actinopycnidium (Krassilnikov, 1962), Micropolyspora (Lechevalier, Solotorovsky, and McDurmont, 1961), and Microellobosporia (Cross, Lechevalier, and Lechevalier, 1963) have shown extensive morpholozical diversities in actinomycete hyphae. According to the conventional classification systems of various authors (Baldacci, 1958; Couch, 1963; Krassilnikov, 1949; Lechevalier et al., 1961; Cross et al., 1963; Nonomura and Ohara, 1957; Waksman, 1961), species of actinomycetes are grouped together in separate genera or families chiefly on the basis of their morphological and ecological characteristics. However, studies by Cummins and Harris (1958), Romano and Nickerson (1956), Romano and Sohler (1956), and Sohler, Romano, and Nickerson (1958) on the cell-wall composition of species in four common genera of actinomycetes, Actinomyces, Micromwnospora, Nocardia, and Streptomyces, have clarified the importance of cell-wall analysis for the characterization of actinomycetes. The present study of the cell-wall composition of many other morphologically distinct actinomycetes was undertaken to examine the relationship, if any, between morphogenetic potentiality and cell-wall composition, and to elucidate the extent of applicability of cell-wall analysis to the taxonomy of this group of organisms. MATERIALS AND METHODS Organisms. The names and sources of the organisms used in this study are listed in Table 1, in which strains are listed by the names under which they were received. Most of the organisms were obtained from their original reporters, and a few were from type culture collections. In the case of Streptomyces sp [= Actinopycnidium elongatum (Nakazawa) Krassilnikov], only a single conidial isolate was used, so that no fruiting body was observed in the culture (see Nakazawa, 1956, 1962, 1964). Most of the strains listed were chosen for the morphological characteristics by which each genus was established. Streptomyces rubrireticuli, S. hachijoensis, and S. netropsis were selected as the representatives of straight or spiral whorl-forming Streptomyces (= Streptoverticillium Baldacci), for which no cell-wall analysis had been reported. Streptomyces griseus ATCC is the strain reported to produce sclerotic granules on Trypticase Soy Agar (Gattani, 1957). S. viridochromo- 444

2 VOL. 89, 1965 CELL-WALL COMPOSITION OF ACTINOMYCETES 445 TABLE 1. Names and sources of strains of actinomycetes used Name of organism No. or name of strain Source Actinoplanes philippinensis... P-15 A. utahensis Actinoplanes sp... E3-15 Actinopycnidium caeruleum... Actinosporangium violaceum... Amorphosporangium auranticolor. Ampullariella digitata... A. regularis... Chainia antibiotica... C. violens... Microbispora amethystogenes... M. rosea... Microellobosporia cinerea... M. flavea... Micromonospora chalcea... USSR RIA-729 USSR RIA USSR RIA-565 M-8 M-20-3* S Micromonospora sp Micropolyspora brevicatena... Promicromonospora citrea... Spirillospora albida... Streptomyces griseus... S. hachijoensis... S. netropsis... S. rubrireticuli W USSR RIA ATCC * H-2609 NRRL S. viridochromogenes... IFO 3113 Streptomyces sp * Streptosporangium album... S. amethystogenes... S. roseum... S. viridialbum... S. vulgare... S-16-1* S-5 27b-1* S-20 S-1, Department of Botany, University of North Carolina H. A. Lechevalier, Institute of Microbiology, Rutgers, The State University N. A. Krassilnikov, Microbiology Institute, Academy of Science USSR, Moscow, USSR N. A. Krassilnikov H. A. Lechevalier N. A. Krassilnikov Y. Ohara, Faculty of Engineering, Yamanashi University, Yamanashi, Japan Y. Ohara H. A. Lechevalier H. A. Lechevalier H. L. Jensen, Department of Bacteriology, State Laboratory of Soil and Crop Research, Lyngby, Denmark (isolated by H. S0rensen) Department of Agricultural Chemistry, University of Tokyo, Tokyo, Japan H. A. Lechevalier N. A. Krassilnikov Nikken Chemical Co., Ltd., Tokyo, Japan Institute of Infectious Diseases, University of Tokyo Northern Utilization Research and Development Division, Peoria, Ill. Department of Agricultural Chemistry, University of Tokyo Institute of Fermentation, Osaka, Japan K. Nakazawa, Takeda Chemical Industries, Ltd., Osaka, Japan Y. Ohara Y. Ohara Y. Ohara Y. Ohara * Single conidial or sporal isolates from the respective parent cultures obtained by use of a micromanipulator. The number after the dash refers to the number of isolations from the respective parent culture. genes is a representative of Streptomyces strains with spiny conidia. Medium and cultural conditions. Unless otherwise stated, the organisms were grown in shake flasks in a medium of the following composition (modified from Romano and Sohler, 1956): dextrose, 10 g; sodium glutamate, 10 g; Difco yeast extract, 3 g; K2HPO4, 1 g; MgSO4-7H20, 0.2 g; CaCl2-2H20, 0.02 g; ZnSO4*7H20, g; FeSO4V 7H20, g; and distilled water, 1,000 ml. CaCl2 was autoclaved separately and added aseptically to each flask. The cultures were incubated at 28 C on a reciprocal shaker at 120 strokes per min. The time of incubation varied with each organism because of differences in the rate of growth and in the rapidity of aerial hyphae formation. Generally, they were harvested at late logarithmic phase of growth when no aerial hyphae or morphologically distinct bodies were formed; in other words, undifferentiated vegetative hyphae were used in these experiments. Preparation of cell walls. With the exception of Promicromonospora, all cells were harvested on a Buchner funnel and were washed in distilled water four times; the cells of Promicromonospora were harvested by centrifugation. They were then resuspended in distilled water and broken by sonic oscillation (10 kc). The time of breakage was 10 to 40 min, varying with the organisms studied.

3 446 YAMAGUCHI J. BACTERIOL. Disrupted cells Centrifuged two to several times at 1,000 to 1,800 X g for 20 min Sediment (discarded) Supernatant fluid (discarded) Supernm ttant fluid Sediment Centrifuged at 4,000 to 7,500 X g for 30 min Repeatedly washed with water, then with 1 M NaCl solution, and finally with water Purified wall Trypsin and pepsin digestions; lyophilized Crude wall Lyophilized Extracted with 0.5% KOH in ethanol for 48 hr at 37 C; trypsin and pepsin digestions; lyophilized Alkaline ethanol-treated purified wall FIG. 1. Procedure used for the preparation of cell walls. The degree of breakage was checked by phase microscopy. After the breaking of the cells, the cell-wall fraction was prepared according to the procedure described in Fig. 1. The general procedures followed were those of Cummins and Harris (1956), with minor modifications; differential centrifugation was done in a cold room at 5 C. The disrupted cell suspension was centrifuged two or more times at 1,000 to 1,800 X g for 20 min. After all unbroken cells were removed, the supernatant fluid was centrifuged at 4,000 to 7,500 X g for 30 min to collect the cell-wall fraction. This fraction was washed twice with distilled water, then three times with 1 M NaCl, and finally was exhaustively washed with distilled water. A part of this fraction was lyophilized and designated as "crude wall." The remainder was resuspended in 0.05 M phosphate buffer (ph 7.6) and was digested with crystalline trypsin (0.5 mg/ml; Nutritional Biochemicals Corp., Cleveland, Ohio) at 37 C overnight in the presence of chloroform. The mixture was then centrifuged; the sediment was washed twice in distilled water, resuspended in 0.02 N HCl with 1 mg/ml of crystalline pepsin (Nutritional Biochemicals Corp.), and digested overnight. After this peptic digestion, the material was finally washed several times with distilled water, lyophilized, and designated as "purified wall." A part of the crude wall was extracted with 0.5% KOH in ethanol for 48 hr at 37 C, washed with neutral ethanol, and then washed several times with distilled water. The material was then digested with trypsin and pepsin (as was the purified wall fraction), lyophilized, and designated as "alkaline ethanol-treated purified wall." Purity of the preparations was checked by nucleic acid analysis and, in some cases, by electron microscopy. These showed that the preparations were adequately pure materials. Paper chromatography of amino acids and amino sugars. Samples of about 15 mg were hydrolyzed in 6 N HCl in sealed ampoules placed in a water bath at 100 C for 8 hr. After cooling, the samples were filtered and evaporated to dryness on a boiling-water bath. They were then redissolved in distilled water, and an amount corresponding to 1 mg of the wall was applied to each paper. In all cases two-dimensional descending chromatograms were prepared, by use of Toyo no. 51 A filter paper. Butanol-pyridine-water (80:80:40)

4 VOL. 89, 1965 CELL-WALL COMPOSITION OF ACTINOMYCETES 447 containing 1% concentrated ammonia was used for the first solvent; phenol-water (500 g:125 ml) containing 1% concentrated ammonia and 8 mg/ 100 ml of 8-hydroxyquinoline, for the second. Papers were run in duplicates. For one paper, the spots were revealed by dipping the chromatogram rapidly through a solution of 0.2% (w/v) ninhydrin in 95% acetone and 5% water, to which about 2% pyridine was added immediately before use. For the other paper, spots were revealed by the Elson-Morgan reaction to confirm the identity of amino sugars. Chromatographic separation of the stereoisomers of diaminopimelic acid (DAP). The chromatographic method described by Hoare and Work (1957) with methanol-water-10 N HCl-pyridine (80:17.5:2.5:10, v/v) was used to separate LLstereoisomer from meso- and DD-stereoisomers. The amount corresponding to 0.3 mg of the wall was applied to the paper. Enzymatic separation of the meso-dap from DD-DAP was not attempted in this experiment. A "slow-moving component" was also noticed during this phase of the work. This substance was reported by Hoare and Work (1957) and others. It gave the same greenish reaction as DAP but moved more slowly than any of the DAP isomers. No further study of this substance was made. Determination of sugars. Samples of about 30 mg were hydrolyzed in 2 N H2SO4 in sealed ampoules at 100 C for 2 hr. After cooling, the hydrolysates were neutralized with hot, saturated Ba(OH)2 and centrifuged, and the supernatant fluids were freeze-dried. The dried residues were desalted by the pyridine method, and the final products were dissolved in distilled water. The amount corresponding to 4 mg of the wall was applied to each paper. Chromatographic procedures were the same as those used for amino acids, except that ammonia was omitted from both solvent systems. The spots were revealed by the reagent mixture containing: aniline, 2.0 ml; phthalic acid, 3.3 g; acetone, 95 ml; and distilled water, 5 ml; this treatment was followed by heating at 105 C for 5 min. RESULTS All substances present in each chromatogram were expressed in relative amounts, according to the relative sizes and intensities of their spots. Depending upon each group of substances, somewhat different grading systems were used (Table 2). Presence or absence of hexosamines and methylpentoses was not mentioned in each case, for glucosamine and muramic acid were detected in every cell-wall preparation, and galactosamine, rhamnose, and fucose in none. Cell-wall composition of various morphologically distinct actinomycetes. Alkaline ethanol-treated purified walls were used for amino acid analysis, and crude walls were used for sugar analysis. The results are arranged in three groups, A, B, and C, for clarity of presentation (Table 2). Ordinary sporangium-forming actinomycetes. In Table 2A, the strains are grouped according to the kind of sporangium-bearing hyphae and the motility of sporangiospores. The first group, comprising the first five strains, produces sporangia on vegetative hyphae and has motile spores. In the second group (Amorphosporangium auranticolor), the sporangium is borne on vegetative hyphae but the spores are nonmotile. The third group (Spirillospora albida) bears its sporangia on aerial hyphae and the spores are motile, and the fourth group (the last seven strains) also has its sporangia on aerial hyphae, but the spores are nonmotile. (Microellobosporia has its sporangium also on vegetative hypha.) Strains of groups one and two do not produce aerial hyphae, whereas those of groups three and four do so. Actinoplanes, Ampullariella, and Amorphosporangium, which are in the first and the second groups and which produce sporangia only on vegetative hyphae, had more or less the same amino acid composition: none to a large amount of the "slow-moving component," meso-dap or DD-DAP, or both, and a smaller amount of LL- DAP, glutamic acid, glycine, and alanine. They had none, or different amounts, of various sugar components, and most of them had arabinose and galactose. Two unidentified spots were observed in the chromatograms of Actinoplanes. An "unidentified pink spot," which was found in A. philippinensis P-15 and Actinoplanes sp. E3-15, as well as in two strains of Micromonospora, moved faster than mannose in butanol-pyridine-water-ammonia but at nearly the same rate in phenol-water-ammonia; it gave a color reaction with aniline hydrogen phthalate similar to that of a pentose. An "unidentified olive spot," which was found only in A. philippinensis P-15, was obtained in the region of N-acetylglucosamine, 2-deoxygalactose, and 2-deoxyribose; however, it occupied a different position from either of them and gave a hexose color reaction with aniline hydrogen phthalate, No further study was made of these substances. The cell-wall composition of Spirillospora albida 761 was different from those of the above strains, containing meso-dap or DD-DAP, or both, and a smaller amount of LL-DAP, glutamic acid, alanine, and galactose. The "slow-moving component" and glycine were not found in appreciable amounts. Strains in the last group of sporangium-forming actinomycetes had different cell-wall compositions according to their genera. Five strains of Streptosporangium had a cell-wall composition similar to the preceding strain and had meso- DAP or DD-DAP, or both, and a smaller amount of LL-DAP, glutamic acid, and alanine as major

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6 l'ol. 89, 1965 CELL-WALL COMPOSITION OF ACTINOMYCETES 449 amino acid components; contents of aspartic acid, serine, glycine, lysine, valine, and leucine varied from none or trace to fair. These strains had little, if any, pentose, and contained no hexoses or various amounts of them. On the other hand, two strains of Microellobosporia had LL- DAP, glutamic acid, glycine, and alanine as major amino acid components; no other amino acid was found in appreciable amount. Sclerotium, pycnidium, or actinosporangiumforming actinomycetes. As shown in Table 2B, three strains of sclerotic granule-forming organisms, Streptomyces griseus ATCC (Gattani, 1957), Chainia antibiotica 3750, and C. violens USSR RIA-565; two strains of pycnidium-type fruiting body-forming organisms, Actinopycnidium caeruleum USSR RIA-729 and Streptomyces sp (Nakazawa, 1956, 1962, 1964) [ = Actinopycnidium elongatum (Nakazawa) Krassilnikov]; and one strain of actinosporangium-forming Actinosporangium were obviously closely related in cell-wall composition, the characteristic components being LL-DAP, glutamic acid, glycine, and alanine, which was the same pattern as that of Microellobosporia. Other actinomycetes. In Table 2C, strains are also arranged according to their morphological characteristics. The first three strains do not produce aerial hyphae or only produce them poorly, and form conidia singly on vegetative hyphae. The vegetative hyphae of Promicromonospora fragment like Nocardia, whereas those of two strains of Micromonospora do not. Micropolyspora brevicatena 1086W produces conidia on both vegetative and aerial hyphae, and its vegetative hyphae have a tendency to fragment. The rest of the strains produce conidia on aerial hyphae, and their vegetative hvphae do not fragfment. The cell wall of Promicromonospora citrea USSR RIA-562 did not have DAP, but had lysine, instead, in addition to glutamic acid, alanine, and hexoses. On the other hand, the cell wall of Micromonospora had a trace to a large amount of the "slow-moving component," meso-dap or DD-DAP, or both, and a smaller amount of LL-DAP, glutamic acid, glycine, and alanine, plus pentoses and various amounts of hexoses. The pattern is similar to that of Actinoplanes. According to Cummins and Harris (1958), cell walls of five strains of Micromonospora had larger amounts of LL-DAP than of other stereoisomers, and had no sugar, whereas Hoare and Work (1957) reported nearly the same amount of LL and other stereoisomers in whole-cell hydrolysates. Cummins and Harris (1958) cultured cells on 1 % glucose digest broth, probably under stationary conditions. As the composition of the glucose digest broth is not mentioned in their paper, I used Waksman's medium (dextrose, 1%; meat extract, 0.5%; peptone, 0.5%; NaCl, 0.5%; ph adjusted to 7.0 to 7.2), cultured MI. chalcea S in it under stationary conditions, and obtained the crude wall. This fraction contained much meso-dap or DD-DAP, or both, but no sugar. Micropolyspora brevicatena 1086W cell walls had meso-dap or DD-DAP, or both, glutamic acid, and alanine as major amino acid components, and trace amounts of aspartic acid, serine, glycine, valine, and leucine. These walls also contained large amounts of arabinose and galactose, and small amounts of other sugars. Cell walls of two strains of Microbispora had meso-dap or DD-DAP, or both, and smaller amounts of LL-DAP, glutamic acid, and alanine as major amino acid components; aspartic acid, serine, and glycine contents of these walls varied from none to trace amounts; in addition, they contained small amounts of sugars. This pattern is similar to, or the same as, that of Spirillospora or Streptosporangium. Cell walls of four strains of Streptomyces, including the three whorl-formers, had LL-DAP, glutamic acid, glycine, alanine, and no, to small amounts of, sugars. They had no, or little, pentose. This pattern is the same as those of Microellobosporia and others. Difference in patterns of cell-wall amino acid composition depending upon the difference in treatment. In most cases, the cell-wall patterns were essentially the same whether crude wall, purified wall, or alkaline ethanol-treated purified wall was used. In the following cases, however, differences were noticed, as shown in Table 3. Crude wall and purified wall of seven strains belonging to Streptosporangium or Microbispora showed much more complex amino acid patterns than did alkaline ethanol-treated purified wall. They had more aspartic acid, serine, glycine, threonine, lysine, valine, tyrosine, and leucine. Purified wall of these strains had only slightly less aspartic acid and lysine than the crude wall, and mere alkaline ethanol extraction of the crude wall did not simplify the amino acid pattern. Thus, alkaline ethanol extraction before the enzyme treatments seemed to be necessary. Walls of Micropolyspora showed a similar tendency. Crude wall and purified wall of three whorlforming Streptomyces (= Streptoverticillium Baldacci), S. hachijoensis H-2609, S. netropsis NRRL 2268, and S. rubrireticuli 3631, had lysine, in addition, as a minor component. DISCUSSION Cell-wall composition of various genera of actinomycetes. From the preceding results it is clear that,

7 450 YAMAGUCHI J. BACTERIOL. TABLE 3. Difference in patterns of cell-wall amino acid composition depending upon the difference in cell-wall treatment* Organism Fraction 0 * Serine Glycine * si Lysine 0~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Streptospor- Crude wall, angium and Purified wall Microbi- Alkaline etha- TR- TR- - --TR -_ spora (7 nol-treated strains) purified wall Micropoly- Crude wall TR TR - spora (1 Purified wall TR - strain) Alkaline etha- TR TR TR - TR - TR nol-treated purified wall Whorl-form- Crude wall _ ing Strep- Purified wall _ tomyces (3 Alkaline etha- - _ _ strains) nol-treated purified wall * For the grading systems of each component, see Table 2. as previous workers have suggested, all genera of actinomycetes have the same general type of cellwall composition as gram-positive bacteria, having three or four amino acids, glucosamine, muramic acid, and sugars if alkaline ethanoltreated purified walls are used for amino acid analysis. These genera may be placed in at least five groups on the basis of cell-wall composition; the characteristic components and the genera belonging to each group are presented in Table 4. If crude walls or purified walls are compared, walls of Streptosporangium, Microbispora, and Micropolyspora show the most complex amino acid composition; walls of Streptoterticillium have lysine, in addition, as a minor component (Table 3). Promicromonospora, an aerobic, soil form of actinomycete, has the same type of cell-wall composition as the morphologically different, anaerobic or microaerophilic, animal form of Actinomyces (A. israelii). Micropolyspora has the Nocardia type of cell-wall, though it had a more complex amino acid pattern in crude wall or purified wall. Genera as different, under the present taxonomic criteria, as Actinoplanes and Mlkicromonospora, Spirillospora and Microbispora, or Streptomyces and Actinopycnidium have the same, or a similar, type of cell-wall composition. These points will be discussed in more detail below. Cummins and Harris (1956) reported that cell-wall composition is a stable character, unaffected by cultural conditions such as medium and growth conditions. In the present investigation, only the genera Streptomyces and Micromonospora can be compared with the results of Hoare and Work (1957), Cummins and Harris (1958), Romano and Nickerson (1956), Romano and Sohler (1956), Sohler et al. (1958), Davis and Baird-Parker (1959), and others. The cellwall composition of Streptomyces was essentially the same as that reported by these workers, whereas that of Micromonospora differed as stated above. In the present work, all strains were cultured under the same experimental conditions one or more times unless otherwise specified. Under other experimental conditions, characteristic cell-wall composition might be somewhat different from that shown in Table 4. Morphogenetic potentiality of the actinomycete hyphae and cell-wall composition. In the present experiments, undifferentiated vegetative hyphae were used for comparisons, and they were found different in cell-wall composition (Table 4). It is clear that organisms of the same cell-wall composition have some morphological properties in common. For instance, organisms of groups 1 and 2 all have fragmenting vegetative hyphae, and do not produce conidia on aerial hyphae, except for some strains of Nocardia and Micropolyspora; organisms of group 3 do not produce aerial hyphae; the morphological characteristic of strains of group 4 is only on aerial hyphae; and some organisms of group 5 produce various kinds

8 VOL. 89, 1965 CELL-WALL COMPOSITION OF ACTINOMYCETES 451 Group TABLE 4. Characteristic cell-wall components of various genera of actinomycetes Characteristic cell-wall components Genera Amino acids Sugars 1. Actinomyces Actinomyces,* Promicromono- Lysine, glutamic acid, ala- Galactose, no pentype spora nine tose 2. Nocardia type (Mycobacterium), (Coryne- Meso- and/or DD-DAP, glu- Arabinose, galactose bacterium), Nocardia,* Mi- tamic acid, alanine cropolysporat 3. Actinoplanes Actinoplanes, Ampullariella, Meso- and/or DD-DAP and May or may not have type Amorphosporangium, Mi- smaller amount of LL-DAP, pentoses cromonospora glycine, glutamic acid, alanine; may or may not have "slow-moving component" 4. Streptosporan- Spirillospora, Streptosporan- Meso- and/or DD-DAP and No or little pentose, gium type gium,t Microbisporat (= smaller amount of LL-DAP, no characteristic Waksmania) glutamic acid, alanine sugar 5. Streptomyces Streptomyces, Streptoverticil- LL-DAP, glycine, glutamic No or little pentose, type lium,t Chainia, Microellob- acid, alanine no characteristic osporia, Actinosporangium, sugar Actinopycnidium * From Cummins and Harris (1958). t If crude walls or purified walls are compared, walls of Micropolyspora, Microbispora, and Streptosporangium have by far the most complex amino acid pattern, and walls of Streptoverticillium have lysine, in addition, as a minor component, as shown in Table 3. of bodies, such as sclerotic granules, pycnidiumlike fruiting bodies. or others. Taxonomic criteria, such as production of motile or nonmotile spores, of monospores, bispores, or sporangium, seem to be of less importance than cell-wall composition in discussing relationships among genera. It seems reasonable to consider that each genus in the same cell-wall group may represent a series of morphological differentiations in vegetative or aerial hyphae, or both. For instance, group 3 organisms may show a series of morphological differentiations in vegetative hyphae of micromonospore-formersporangium-former-zoosporangium-former. Phylogenetic consideration. From this line of reasoning, it follows that the morphological differentiation in various genera of actinomycetes does not seem to have proceeded along a single line of development, but seems to have occurred separately and often in parallel in various otherwise related groups of organisms, for instance, among those having the same cell-wall pattern. In addition, I deem it more reasonable to consider that the morphological differentiation in the vegetative hyphae proceeds separately or sometimes side by side with that in the aerial hyphae. A tentative and diagrammatic representation showing the relative position of various genera of actinomycetes, based on cell-wall composition and morphological differentiations, is shown in Fig. 2. In the figure, genera are grouped according to cell-wall composition, and, within a group, each genus is laterally arranged according to the superficial degree of morphological differentiation. Where differentiations in both aerial and vegetative hyphae are recognized, only that of heading of each column is shown in the diagram. In the ordinate, each group is placed according to lysine or DAP-stereoisomer component following the reverse of the biosynthetic pathway of lysine, for ease of understanding. Possible intergroup relationships and those with bacteria are not mentioned in the diagram, for they seem to be polyphyletically connected. Additional similar studies of thermophilic actinomycetes, as well as of new morphological types of organisms which may be isolated in the future, will greatly clarify the situation. For discussions of the phylogeny of actinomycetes by other authors, refer to Jensen (1953), Bisset (1959), Hesseltine (1960), Jones and Bradley (1962, 1964), and others. Cell-wall analysis and taxonomy of actinomycetes. It is quite obvious that cell-wall analysis is an invaluable tool for the taxonomy of this group of

9 452 YAMAGUCHI J. BACTERIOL. Differentiation in vegetative hyphae Lysine Act- Pm Meso- and/ soft hard - DAP Noc Noc \ DAP~~~~~P lalp Differentiation in aerial hyphae Meso- and/ or DD- Apl Mb-Ssp DAP Mm-AmsA Spa plus LL- Sp-s Aml DAP LL-DAP Sv Sm-AMe Ch,As Apyc FIG. 2. Tentative and diagrammatic representation showing relative positions of various genera of mesophilic actinomycetes, based upon cell-wall composition and morphological differentiation. (Where differentiations in both aerial and vegetative hyphae are recognized, only that of heading of each column is shown in the diagram.) Abbreviations used for generic names: Act, Actinomyces; Apl, Actinoplanes; Apyc, Actinopycnidium; As, Actinosporangium; Ams, Amorphosporangium; Aml, Ampullariella; Ch, Chainia; Mb, Microbispora (= Waksmania); Me, Microellobosporia; Mp, Micropolyspora; Mm, Micromonospora; Noc, Nocardia; Pm, Promicromonospora; Sps, Spirillospora; Sm, Streptomyces; Ssp, Streptosporangium; Sv, Streptoverticillium. organisms. And, from the foregoing results and discussions, it is also apparent that the conventional classification systems, based primarily upon morphological characteristics, at least at the genus or family level, deviate very much from the classification by cell-wall composition; for instance, genera belonging to Actinoplanaceae had three different types of cell-wall pattern. Yet, it is still premature to decide which method is more logical, for either system has its own advantages and disadvantages taxonomically. Generally, cell-wall analysis is more troublesome to taxonomists and requires much work in comparing a large number of organisms. In spite of this, it is strongly hoped that cell-wall composition, as well as some other useful analysis of the biochemical structure of organisms, will be used to establish a genus or family for new isolates, or to try to recognize phylogenetic relationships among actinomycetes. These relationships often give decisive evidence that a genus or a family is exclusively independent from or inclusively related to otherwise different organisms. Some comments about the placement of certain genera or species follows. Promicromonospora. Krassilnikov et al. (1961) reported that this organism resembled Micromonospora by its single, terminal spore formation on the vegetative hyphae, and that it resembled Proactinomyces (Nocardia) by its segmentation of the vegetative hyphae. However, its cell-wall analysis did not resemble either of them but resembled Actinomyces, which is quite a different genus according to the conventional taxonomic criteria, and so seems to represent a separate group of organisms just as he had placed it. Micropolyspora. This genus, which was described by Lechevalier et al (1961), had the nocardial type of wall, though it had a more complex amino acid pattern in crude wall or purified wall. In fact, Micropolyspora has many characters in common with hard, conidia-forming Nocardia, such as partial acid-fastness, fragmentation of the vegetative hyphae, and others, in addition to the cell-wall composition. Thus, it seems more reasonable to place Micropolyspora and Nocardia in the same group or family in actinomycete classification. Streptoverticillium. Baldacci (1958) proposed a new generic name, Streptoverticilliurn, for whorlforming streptomycetes. These organisms have been recognized by many as a distinct species of Streptomyces, chiefly for their morphological peculiarities and some physiological characters. It is interesting to note that these organisms, whether they form straight or spiral whorls, have slightly different cell-wall compositions, although the characteristic component is not the one in their mucopeptide. This fact, together with the finding by Jones and Bradley (1962) of actinophage susceptibility, strengthens the view of Baldacci (1958), who placed whorl-formers in a separate genus. Chainia. The chief distinguishing character of the genus Chainia is the formation of spherical, sclerotic granules (Thirumalachar, 1955). However, Gattani (1957) had found that even Streptomyces griseus would form these structures under certain cultural conditions, and concluded that there was no justification for creating a new genus on the basis of these structures. From cellwall analysis, no additional evidence favoring the separation of Chainia from Streptomyces was found. Actinopycnidium. A somewhat similar situation exists in the genus Actinopycnidium. This genus was created by Krassilnikov (1962) for the actinomycetes which were characterized by the formation of a fruiting body of the pycnide type. On the other hand, Nakazawa (1956, 1962, 1964), who had precisely described this kind of or-

10 170L. 89, 1965 CELL-WALL COMPOSITION OF ACTINOMYCETES ganism (Streptomyces sp. 5866) earlier than Krassilnikov, has insisted that his organism is really a Streptomyces; he theorized that some of the streptomycetes would perform, besides the hitherto known asexual life cycle, a sexual life cycle, in which this kind of structure is formed. In this case, also, no decisive evidence was obtained from the cell-wall analvsis. ACKNOWLEDGMENTS I gratefully acknowledge the kind generosity of the many investigators who sent cultures. In addition, I wish to thank H. lizuka for his continued encouragement. LITERATURE CITED BALDACCI, E Development in the classification of actinomycetes. Giorn. Microbiol. 6: BISSET, K. A The morphology and natural relationships of saprophytie actinomycetes. Progr. Ind. Microbiol. 1: COUCH, J. N Some new genera and species of the Actinoplanaceae. J. Elisha Mitchell Sci. Soc. 79: COUCH, J. N A proposal to replace the name Ampullaria Couch with Ampullariella. J. Elisha Mitchell Sci. Soc. 80:29. CROSS, T., M. P. LECHEVALIER, AND H. LECHE- VALIER A new genus of the Actinomycetales: Microellobosporia gen. nov. J. Gen. Microbiol. 31: CUMMINS, C. S., AND H. HARRIS The chemical composition of the cell wall in some grampositive bacteria and its possible value as a taxonomic character. J. Gen. Microbiol. 14: CUMMINS, C. S., AND H. HARRIS Studies on the cell-wall composition and taxonomy of Actinomyeetales and related groups. J. Gen. Microbiol. 18: DAVIs, G. H. G., AND A. C. BAIRD-PARKER The classification of certain filamentous bacteria with respect to their chemical composition. J. Gen. Microbiol. 21: GATTANI, M. L Production of sclerotic granules by Streptomyces sp. Nature 180: HESSELTINE, C. W Relationships of the Actinomyeetales. Mycologia 52: HOARE, D. S., AND E. WORK The stereoisomers of a,e-diaminopimelic acid. 2. Their distribution in the bacterial order Actinomycetales and in certain Eubacteriales. Biochem. J. 65: JENSEN, H. L The genus C\:Aocardia) (or Proactinomyces) and its separation from other ((Actinomyeetales)), with some reflections on the 453 phylogeny of the Actinomycetes. Intern. Congr. Microbiol., 6th, Rome, p JONES, L. A., AND S. G. BRADLEY Relationship of Streptoverticillium and Jensenia to other actinomycetes. Develop. Ind. Microbiol. 3: JONES, L. A., AND S. G. BRADLEY Relationships among Streptomycetes, Nocardiae, Mycobacteria and other Actinomycetes. Mycologia 56: KRASSILNIKOV, N. A Guide to the identification of bacteria and actinomycetes. Akademiya Nauk SSSR, Moscow. KRASSILNIKOV, N. A A new Actinomyces genus Actinopycnidium n. gen. of the Actinomycetaceae family. Mikrobiologiya 31: KRASSILNIKOV, N. A., L. V. KALAKOUTSKII, AND N. F. KIRILLOVA A new genus of Actinomycetales Promicromonospora gen. nov. Izv. Akad. Nauk SSSR Ser. Biol., p KRASSILNIKOV, N. A., AND J. TSI-SHEN Actinosporangium-a new genus of the Actinoplanaceae family. Izv. Akad. Nauk SSSR Ser. Biol., p LECHEVALIER, H. A., M. SOLOTOROVSKY, AND C. I. MCDURMONT A new genus of the Actinomycetales: Micropolyspora gen. nov. J. Gen. Microbiol. 26: NAKAZAWA, K Some observation on the reproduction of Streptomyces No J. Agr. Chem. Soc. Japan 30: NAKAZAWA, K On the sexuality of a species of streptomycetes. Trans. Japan. Mycol. Soc. 4:9-10. NAKAZAWA, K Studies on the life cycle of Streptomyces No Trans. Japan. Mycol. Soc. 4: NONOMURA, H., AND Y. OHARA Distribution of actinomycetes in the soil (II). Microbispora, a new genus of Streptomycetaceae. J. Fermentation Technol. 35: ROMANO, A. H., AND W. J. NICKERSON The biochemistry of the Actinomycetales. Studies on the cell wall of Streptomyces fradiae. J. Bacteriol. 72: ROMANO, A. H., AND A. SOHLER Biochemistry of the Actinomyeetales. II. A comparison of the cell wall composition of species of the genera Streptomyces and Nocardia. J. Bacteriol. 72: SOHLER, A., A. H. ROMANO, AND W. J. NICKERSON Biochemistry of the Actinomycetales. III. Cell wall composition and the action of lysozyme upon cells and cell walls of the Actinomycetales. J. Bacteriol. 75: THIRUMALACHAR, M. J Chainia, a new genus of the Actinomycetales. Nature 176: WAKSMAN, S. A The actinomycetes, vol. 2. Classification, identification and descriptions of genera and species. The Williams & Wilkins Co., Baltimore.