Cell Wall Composition in Relation to the Taxonomy of Some Actinoplanaceae'

Transcription

1 JOURNAL OF BACTERIOLOGY, Dec. 1967, p Copyright 1967 American Society for Microbiology Vol. 94, No. 6 Printed in U.S.A. Cell Wall Composition in Relation to the Taxonomy of Some Actinoplanaceae' PAUL J. SZANISZLO2 AND HARRY GOODER Department of Botany and Department of Bacteriology, University of North Carolina, Chapel Hill, North Carolina Received for publication 11 September 1967 Hydrolytic residues of the cell walls of 48 strains of Actinoplanaceae, previously assigned to 10 species and the four genera, Actinoplanes, Ampullariella, Amorphosporangium, and Pilimelia, were examined by paper chromatography and column chromatography. Comparisons were made for taxonomic purposes between the groupings obtained, by use of chemical characters and the groupings currently recognized morphologically. Most of the species investigated had qualitatively distinct cell wall compositions. Often, however, the cell wall compositions of species in different genera were more similar, in some respects, than were those of species in the same genus. Quantification of the cell wall amino acids and amino sugars substantiated that cross-generic similarities existed. Based on these results and the morphological conclusions reached by other investigators, a single-genus concept is suggested for the Actinoplanaceae examined. Existing schemes of bacterial classification often need re-examination to determine whether traditional criteria are obscuring natural relationships. Among the Actinoplanaceae (Actinomycetales), many species are placed in their genus category on the basis of a relatively few morphological characters. This often necessitates classifying similar Actinoplanaceae into different genera. The most notable examples occur among the species which are morphologically and culturally very similar to Actinoplanes philippinensis, the species on which the family Actinoplanaceae is based. These species are classified among three genera predominantly on the basis of sporangiospore morphology (5, 6). Species producing sporangiospores morphologically similar to those produced by Actinoplanes philippinensis (subspherical with a tuft of polar flagella) are placed in the genus Actinoplanes. Species producing rodshaped sporangiospores with a tuft of polar flagella are placed in the genus Ampullariella, and species producing rod-shaped sporangiospores with no flagella are placed in the genus Amorphosporangium. 1 Part of a dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Botany. 2 Present address: Laboratory of Applied Microbiology, Division of Engineering and Applied Physics, Harvard University, Cambridge, Mass The present investigation was undertaken to compare the cell wall composition of numerous strains of Actinoplanes, Ampullariella, and Amorphosporangium to determine whether the traditional criteria are obscuring natural relationships among species presently classified in these genera. Three strains of keratinophilic Actinoplanaceae were also examined. Couch (5) speculated that such Actinoplanaceae probably belong in the genus Ampullariella. However, Kane (12) reported that morphological and cultural differences warranted placing the keratinophilic strains she studied in a new genus which she named Pilimelia. While this investigation was in progress, Yamaguchi (23) and Becker, Lechevalier, and Lechevalier (1) reported on the cell wall composition of various aerobic actinomycetes. Their investigations revealed that strains of Actinoplanes, Ampullariella, and Amorphosporangium had similar, but not necessarily identical, cell wall compositions. However, the investigations of Yamaguchi and Becker et al. did not involve sufficient numbers of these Actinoplanaceae to allow for extensive speculation on the validity of their classification among three genera. MATERIALS AND METHODS Organisms. The name, number, and general location of the original collection site of each laboratory stock strain examined are listed in Table 1. The strains of Actinoplanes, Ampullariella, and Amorphosporan- 2037

2 2038 SZANISZLO AND GOODER J. BACTERIOL. TABLE 1. Name, number, and general location of original collection site of each strain investigated Actinoplanes Organism Strain no. Collection site philippinensis... Actinoplanes utahensis... Actinoplanes missouriensis... Ampullariella Ampullariella regularis... digitata... Ampullariella lobata... Ampullariella campanulata... Ampullariella sp... Amorphosporangium auranticolor. Pilimelia anulata... Pilimelia terevasa... Pilimelia sp... I ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 2 Philippine Islands 258 Salt Lake City, Utah 259 Salt Lake City, Utah 260 Salt Lake City, Utah 261 Dunphy, Nev. 221 Palonio Pass, Calif. 222 Sierra Nevada Pass, Calif. 263 Dunphy, Nev. 267 Rainbow Valley, Wyo. 431 Hamilton, Mo. 443 Cameron, Mo. 657 North Bend, Ore. 825 Checotale, Okla. 28 Chapel Hill, N. C. 79 Madison, Wis. 154 Clemson, S. C. 164 Grenada County, Miss. 168 Grenada County, Miss. 312 Draper, Va. 395 Indianapolis, Ind. 850 Chapel Hill, N. C. 915 Joyce Kilmer National Park 33 Cheboygan, Mich. 71 Madison, Wis. 118 Chapel Hill, N. C. 131 Chapel Hill, N. C. 137 Hindhead, England 370 Chillicothe, Ohio 386 Indianapolis, Ind. 399 Indianapolis, Ind. 72 Madison, Wis. 74 Madison, Wis. 337 Draper, Va. 65 Douglas, Kan. 126 Chapel Hill, N. C. 151 Chapel Hill, N. C. 182 Durham County, N. C. 640 Tahiti 641 Tahiti 642 Tahiti 643 Borabora 1492 Bombay, India 1539 Mahabalipuram, India 253 Dunphy, Nev. 262 Dunphy, Nev. 11 Walkerton, Ind. Walkerton, Ind Hyderabad, India gium were identified by J. N. Couch (3, 4, 5, 6), and stock cultures of each strain are maintained in his laboratory on Czapek agar (Difco) and peptone- Czapek agar (Czapek agar fortified with 5 g/l,000 ml of Difco peptone). The strains of Pilimelia were identified by W. D. Kane (12) and are maintained by her on peptone-czapek agar. Medium and culture method. The organisms from 3-week-old slants were prepared for inoculation into 1,000 ml of Czapek Dox broth (Difco), fortified with 5 g of peptone (Difco), housed in 1,500-ml low-form flasks, by suspending adequate amounts of each strain in 50 ml of sterile broth and blending for 60 sec at high speed in a cold Waring Blendor micro-cup. After inoculation, the cultures were grown for 5 days at 24 to 26 C with continuous agitation (100 strokes/ min) on a gyratory shaker (model G-10, New Brunswick Scientific Co., New Brunswick, N.J.). The re-

3 VOL. 94, 1967 CELL WALL COMPOSITION IN ACTINOPLANACEAE 2039 sulting vegetative hyphae were harvested by centrifugation at 3,000 X g and washed three times with distilled water. Preparation of cell walls. The hyphae were mechanically disrupted in a Braun homogenizer by the method of Bleiweis, Karakawa, and Krause (2). Disrupted suspensions were centrifuged at 10,000 X g for 10 min. Hyphae with any remaining intact cells in their length formed a tightly packed, colored layer at the bottom of the centrifuge tube. Completely disrupted hyphae were devoid of color and formed a loosely packed, upper white layer, which was rinsed away from the colored layer by repeated washes with distilled water. The cell walls were collected by centrifugation, treated for two periods of 1 hr each in 1% sodium lauryl sulfate, rewashed thoroughly in distilled water, and lyophilized. Enzyme treatment of walls. Lyophilized walls in 100-mg amounts were treated with 10 mg of crystalline ribonuclease in 10 ml of 0.1 M phosphate buffer (ph 7.8) for 3 hr at 37 C, then 5 mg of crystalline trypsin was added, and incubation was continued for 3 hr at 37 C. The walls were deposited by centrifugation, washed three times with distilled water, suspended in 20 ml of 0.2 N HCI containing crystalline pepsin (1 mg/ml), and incubated overnight at 37 C. The walls were collected by centrifugation, thoroughly washed with distilled water, lyophilized, and used in all subsequent analyses. Preparation of samples for chromatography. Paper chromatographic analysis of the walls of each strain for amino acids and amino sugars was conducted after hydrolysis of 10-mg samples of lyophilized walls in 5 ml of 6 N HCI in sealed Pyrex tubes for 16 hr at 100 C. For cell wall sugars, the hydrolysis conditions were 20 mg of lyophilized walls in 2 ml of 2 N H2SO4 in sealed Pyrex tubes for 2 hr at 100 C. Subsequent handling of the hydrolysates was substantially as described by Michel and Gooder (15). Residues from the HCI hydrolysates and H2SO4 hydrolysates were dissolved in 0.4 ml and 0.25 ml of distilled water, respectively. Cell wall samples in 5- mg amounts were prepared for quantitative chromatography as described by Moore and Stein (16). Paper chromatography. Two-dimensional descending paper chromatography was conducted on Whatman no. 1 filter paper, with n-butanol, acetic acid, and water (BAW; 60:10:20, v/v) as the first solvent and 2,6-lutidine (Practical, Matheson, Coleman, and Bell, Cincinnati, Ohio) and water (LW; 65:35, v/v) as the second solvent. For amino acids and amino sugars, the hydrolytic equivalent of 0.5 or 1.0 mg of cell walls was applied to the paper, which was subsequently developed for 23 hr with BAW and 28 hr with LW. Spots were revealed with ninhydrin. For sugars, the hydrolytic equivalent of 0.8 mg and 1.6 mg of walls was applied to papers which were developed for 30 hr with BAW and 20 hr with LW. Sugar spots were revealed with acid aniline phthalate reagent. All samples were chromatographed at least two times and verification of the components was accomplished, where possible, by using pure substances as markers subjected to identical chromatographic conditions both separately and after admixture with the hydrolytic samples. Galactose and glucose identification. When both galactose and glucose were present together, as in the hydrolysate of walls of Actinoplanes philippinensis strain 2, the identity of each was verified by adding separately and together glucose oxidase (0.5 unit) and galactose oxidase (0.125 unit) to hydrolysate samples containing 0.8-mg equivalent of wall material. Enzyme mixtures were incubated for 2 hr at 37 C, and the entire sample was then chromatographed to test for the disappearance of the respective sugar or sugars. Deoxyhexose identification. Relatively pure samples of the fast-running sugar detected in wall hydrolysates were obtained by chromatographing as a band the combined H2SO4 hydrolysates of 37 mg of cell walls, with BAW as solvent. After the position of the sugar was located with test strips, the band was eluted from the paper and the eluate filtered through a washed filter, 0.45, pore size (Millipore Corp., Bedford, Mass.), and then taken to dryness over P205. Wash water from a blank chromatogram was similarly treated and used as a control. Both residues were taken up in 2 ml of distilled water. Samples of the sugar solution and control were subjected to the Dische spectrophotometric procedure (9) for 6-deoxyhexoses. Various concentrations of similar samples were also chromatographed overnight with rhamnose and fucose, separately and together, with n-butanol, pyridine, and water (6:4:3, v/v) as the solvent (13), in order to determine the R rhamnose (R,h) of the unknown. Diaminopimelic acid identifications. The paper chromatographic separation of 2, 5-diamino-3-hydroxypimelic acid (HDAPA) and the stereoisomers of 2, 6-diaminopimelic acid (DAPA) was accomplished with the general method of Hoare and Work (10), but with the solvent suggested by Perkins (18). The equivalent of 0.5 mg of cell walls of each strain was chromatographed. Quantitative amino acid analysis. Quantitative chromatography was conducted using a Beckman model 120 amino acid analyzer. The equivalent of 1 mg of HCl-hydrolyzed cell walls was examined, and duplicate determinations were made for the acid and neutral amino acids; however, since major amounts of the basic amino acids were not detected, only single runs were made for their quantification. Electron microscopy. Electron microscope preparations were made by the method of Sharp (19). RESULTS Criteria Of purity. The criteria for purity of all wall fractions were: absence of color in disrupted hyphae packed by centrifugation, and absence of large amounts of particulate matter inside the disrupted cells when examined with the electron microscope. Wall fractions obtained from the initial disruption of hyphae, when examined with the electron microscope, rarely exhibited hyphae with intact cells. Small amounts of particulate

4 2040 SZANISZLO AND GOODER J. BACTERIOL. matter were noticed along the lengths of some hyphae (Fig. 1). Examination of the enzymetreated cell walls showed that most of this material was removed during the enzymatic digestions (Fig. 2). Qualitative cell wlall analyses. Cell wall preparations were made from 48 strains of Actinoplanaceae previously assigned to 10 species and the four genera Actinoplanes, Ampullariella, Amorphosporangium, and Pilimelia. Hydrolytic residues of these cell walls, when subjected to paper chromatography, revealed the substances documented in Table 2-5. The sugars and DAP acids are expressed in relative amounts according to the sizes and intensities of their spots on paper. The relative amounts of glucosamine, muramic acid, glycine, and alanine are not shown because all of these compounds were present in large amounts (4 or greater) in each hydrolytic sample. The grouping of the organisms is presented so as to facilitate discussion, and does not imply relatedness. Strains of Actinoplanes philippinensis, Amorphosporangium auranticolor, and Ampullariella regularis are listed in Table 2. All strains of these morphologically determined species, except strain 28 and strain 312, exhibited nearly identical cell wall sugar patterns. Actinoplanes philippinensis was distinguished from the other two species by not having HDAPA in its cell walls. In contrast, the strains of Amorphosporangium auranticolor were distinguished by having both HDAPA and DAPA in their cell walls, and the strains of Ampullariella regularis were distinguished by not having DAPA in their cell walls. Of the two strains of A. regularis (strains 28 and 312) that differed in their general cell wall sugar constituents, only strain 28 differed extensively, showing simply xylose and the deoxyhexose. The absence of galactose in the cell walls of this organism made it unique among the 48 strains investigated. Yamaguchi (23) analyzed the cell walls of three of the strains listed in Table 2, while Becker et al. (1) analyzed one. Actinoplanes philippinensis strain 2 was reported by Yamaguchi to have the cell wall sugars galactose, arabinose, and mannose, as well as two sugars he was not able to identify. These two unidentified sugars are probably the sugars identified as xylose and 6-deoxyhexose in this work. Amorphosporangium auranticolor strain 253 was examined by both Yamaguchi and Becker et al. Yamaguchi did not detect xylose or the 6-deoxyhexose in the walls of this strain, but Becker et al. reported that this strain had no sugar in major amounts. The third strain examined by Yamaguchi was Ampullariella regularis strain 28. He reported that this strain had no sugars. Analyses of the cell walls of the strains of Actinoplanes utahensis and A. missouriensis revealed somewhat different patterns (Table 3). The distinguishing cell wall components for A. utahensis are HDAPA, galactose, and mannose, and those for A. missouriensis are galactose, glucose, and either HDAPA or both HDAPA and DAPA. Various other components were detected in low concentrations. The strains of A. utahensis usually showed traces of xylose and the 6-deoxyhexose. In contrast, the strains of A. missouriensis showed traces of mannose, arabinose, and xylose. Strain 267 had a cell wall composition more like A. utahensis than A. missouriensis. Results for the strains of A. missouriensis indicated that these strains are in reality a heterogeneous group composed of two chemically distinct subgroups. Cell walls of strain 260 were reported by Yamaguchi to contain galactose, arabinose, and major amounts of both HDAPA and DAPA. Table 4 lists results for strains of Pilimelia and Ampullariella digitata. The three strains of Pilimelia were distinguished by having cell walls containing DAPA, galactose, glucose, mannose, arabinose, and xylose. Those of Ampullariella digitata had cell walls containing HDAPA and all or some of the same sugars. These latter strains showed more variation than was found in any other morphologically determined group. Strain 33 was also studied by Yamaguchi. He similarly reported only galactose and the pink spot (xylose) in this strain. The strains of Pilimelia are interesting because they were the only strains that showed minor amounts of galactose. In addition, they were the only strains, besides Actinoplanes philippinensis strain 2, which exhibited major amounts of DAPA in the absence of HDAPA. Chemically, the Pilimelia sp. seemed more like P. anulata than P. terivasa. The results obtained for Ampullariella lobata and Ampullariella campanulata revealed that these organisms are easily distinguished from all the others (Table 5). Only the cell walls of these strains contained the heptose, small amounts of galactosamine, and in some instances an unknown (described simply as the spot near xylose). In addition, cell walls of all the strains contained galactose and mannose in large amounts. ace amounts of other components were sometimes detected. Two of the three strains designated Ampullariella lobata could be distinguished from the remaining strains by the absence of DAPA. The unidentified strains of Ampullariella seemed clearly to belong in this Ampullariella lobata and A. campanulata complex of organisms. Deoxyhexose. Deoxyhexose, first detected in the cell walls of Actinoplanes philippinensis, gave a

5 VOL. 94, 1967 CELL WALL COMPOSITION IN ACTINOPLANACEAE 2041 FIG. 1. Electron micrograph of disrupted vegetative hyphae which have beeii washed for 2 periods of I hr each in Ic1 sodium lauryl sulfate solution and theen shadowed with platinum-palladium. Note particulate matter along the length of some hyphae. X 25,000. FIG. 2. Electron micrograph of disrupted vegetative hyphae which have heen washed with sodium lauryl sulfate, enzyme-treated with ribonuclease, pepsin, and trypsin, and then shadowed with platinum-palladium. Note absence ofparticulate matter. X 25,000.

6 2042 SZANISZLO AND GOODER J. BACTERIOL. brown color when sprayed with acid aniline phthalate reagent and was thought to be rhamnose. However, when authentic rhamnose was added to the sugar residues before chromatography, the resulting sugar spot became abnormally elongated. Reaction of the isolated unknown sugar with cysteine-sulfuric acid (9) revealed that the chromophore formed resembled that formed by rhamnose and fucose with an absorption maximum at 400 m,u. All of the six other 6-deoxyhexoses are reported to resemble rhamnose and fucose in this test (14). Paper chromatography of the unknown sugar, fucose, and rhamnose showed that the (Rrh) of the unknown (0.96 to 0.97) compared favorably with the Rrh reported for 6-deoxy-L-glucose (13). The Rrh for fucose also compared favorably with TABLE 2. Organism (morphological designation) A. philippinensis... A. auranticolor... A. regularis... the Rrh previously reported for fucose (13), indicating that the different values could be accurately compared. These results indicate that the sugar is 6-deoxy-L-glucose; however, sufficient quantities of the sugar are as yet unavailable for a more definite identification. Galactose and glucose. Cell wall hydrolytic residues from Actinoplanes philippinensis had two components corresponding to glucose and galactose. Residues treated with glucose oxidase showed a single hexose spot corresponding to galactose after chromatography, and residues treated with galactose oxidase showed a single hexose spot corresponding to glucose after chromatography. Residues treated with both enzymes showed no spots corresponding to either glucose or galactose after chromatography. Components in cell walls of Actinoplanes philippinensis, Amorphosporangium auranticolor, and Ampullariella regularisa Strain no DAPA IHDAPA Galactosamine Glucose Xylose ± Spot near xylose - All contain major amounts of glucosamine, muramic acid, glutamic acid, glycine, and alanine. Components graded:,,, (ace). TABLE 3. Organism (morphological designation) A. utahensis... A. missouriensis... Components in cell walls of Actinoplanes utahensis and A. missouriensisa Strain no DAPA HDAPA Heptose Galactose Mannose Arabinose Deoxyhexose Galactosamine Heptose Galac- Glucose Man- Arabiitose Inose Inose Xylose Spot near xylose Deoxyhexose a All contain major amounts of glucosamine, muramic acid, glutamic acid, glycine, and alanine. Components graded:,,, (ace).

7 VOL. 94, 1967 CELL WALL COMPOSITION IN ACTINOPLANACEAE 2043 TABLE 4. Components in cell walls of Pilimelia terivasa, P. anulata, and Ampullariella digitataa Organism St Galac- Mn Arb-Spot Doy (morphological Strain DAPA HDAPA tos- Heptose Galactose Glucose Man- Arabi- Xylose near Deoxydesignation) no. amine ns noexylose hxs P. terivasa... I P. anulata... I Pilimelia sp A. digitata ± a All contain major amounts of glucosamine, muramic acid, glutamic acid, glycine, and alanine. Components graded:,,, (ace). Organism (morphological designation) A. lobata... A. campaniulata.. Ampulariella sp.. Strain no DAPA HDAPA Heptose Galac- Glucose tose Xylose Spot near xylose TABLE 5. Components in cell walls of Ampullariella lobata and A. campaniulata- Galactosamine Mannose Arabinose Deoxyhexose a All contain major amounts of glucosamine, muramic acid, glutamic acid, glycine, and alanine. Components-graded:,,, (ace). Heptose and the compound with a mobility similar to xylose. The heptose and the compound that had a mobility similar to xylose posed special problems in identification. Both gave hexose (brown) colors with acid aniline phthalate reagent, but did not exhibit RF values similar to the more common hexose sugars. The heptose migrated more slowly than galactose and was at first thought to be a uronic acid. However, when co-chromatographed with authentic glucuronic and galacturonic acid, the compound migrated more slowly and did not produce the red color reported to be produced with p-anisidine phosphate or p-anisidine hydrochloride (11, 17). The color produced with the p-anisidine phosphate was yellow-brown; the color with p-anisidine hydrochloride was violet-brown. Davies (8) reported that some aldoheptoses migrate more slowly than galactose and produce a violet-green coloration with the p-anisidine hydrochloride reagent. On the basis of these facts, this compound was tentatively identified as a heptose sugar. The compound that had a mobility similar to xylose gave a brownish color with both acid aniline phthalate reagent and p-anisidine hydrochloride, and is possibly a 2-deoxy sugar or a methylated aldohexose. However, many classes of compounds have constituents exhibiting mobilities similar to this compound. Isomers of 2,6-DAPA. Paper chromatographic separation of the isomers of the 2,6-DAP acids showed that meso-dapa or DD-DAPA, or both, occurred in high concentrations and were the main isomers being detected. It is interesting to note that traces of LL-DAPA were always detected whenever meso- or DD-DAPA, or both,

8 2044 SZANISZLO AND GOODER J. BACTERIOL. were detected in major amounts. Also, traces of meso- or DD-DAPA, or both, were always detected when HDAPA occurred alone. Whenever HDAPA was detected, it was found to occur in high concentrations. Yamaguchi (23) also reported the occurrence of small amounts of the 2,6-DAPA acids in the cell walls of the five previously mentioned organisms that he studied. It seems unlikely that these trace constituents are integral components of the cell wall; they are, in all probability, of cytoplasmic origin. Quantitative cell wall analyses. Results for the amino acid determinations of the walls of the type organisms are presented in Table 6. The values obtained for glucosamine and muramic acid are not included, because they showed more than the 3% maximal variation obtained for the duplicate amino acid determinations; they seemed always to represent a 1 to 1 relationship. Amino acid molar ratios were calculated with glutamic acid as unity. Results indicate that all the type organisms have very similar molar ratios for the amino acids in their cell walls. Glycine is usually present in the largest amount. The amino acids in the cell walls of the two Pilimelia strains appeared to be present in a 1:1:1:1 relationship, and those in Actinoplanes philippinensis appeared to represent a 2:2:1:2 relationship, with alanine being present in the smallest amount. Those strains which did not have DAPA all had somewhat different molar ratios, but such variation could be explained, in most instances, as resulting from small amounts of contaminating amino acids of cytoplasmic origin. The Ampullariella campanulata and Amorphosporangium auranticolor strains possessed significant amounts of cell wall HDAPA and DAPA. The sum total of the two DAP's was only slightly different from the values obtained for the two DAP's when they occurred alone. The Ampullariella regularis strain 28 was also studied quantitatively by Perkins (18). Recalculations of his results showed that he obtained similar values for this organism, even though it was grown and treated in a different way. Thus, all strains of the Actinoplanaceae so far examined contain less alanine than glutamic acid in their cell walls. DISCUSSION The taxonomy of the Actinoplanaceae is based predominantly on morphological characters and a limited number of physiological features. The characters most commonly cited as being important taxonomically are sporangiospore and sporangium shape, sporangiospore flagellation pattern, mode of branching or coiling of the sporogenic hypha, and coloration of cultural media. The results of the present investigation are in accord with the current morphological groupings of the Actinoplanaceae at the species level. However, the same results indicate that these morphological characters may not be adequate, when considered alone, for the classification of Actinoplanaceae at higher levels. This conclusion is reached by comparing results from the qualitative analyses of the sugars, amino sugars, and amino acids found in the vegetative cell walls and the quantitative determinations of the vegetative cell wall amino acids and amino sugars. When these features are considered in combination with the conclusions of Couch (3, 4, 5, 6) and Kane (12), patterns are discerned which suggest that the species examined are more closely related to one another than their present generic positions would indicate. Qualitative analyses of the vegetative cell walls show that every strain examined has basically the same pattern of amino acid and amino sugar constituents. As was also reported by Yamaguchi (23), major amounts of glucosamine, muramic acid, glycine, alanine, glutamic acid, and either meso- or DD-DAPA (or both) and HDAPA (or both) are found in the cell walls of Actinoplanes, Ampullariella, and Amorphosporangium. The present work adds Pilimelia to this list. In contrast to the statement of Cummins and Harris (7), that each genus of gram-positive bacteria appears to have a characteristic pattern of amino acids associated with its cell walls, two genera of Actinoplanaeceae examined in this work have slightly different amino acid patterns among their species. It was observed that, in the cell walls of species of these genera, some species contain DAPA, some species HDAPA, and some species contain both these diaminopimelic acids in addition to the glutamic acid, glycine, and alanine amino acid complement which was present in the cell walls of every strain examined. Whether this variation indicates that the Actinoplanaceae strains previously placed in the same genus but in different species are in reality unrelated, or whether it means that it is not unusual to find various diaminopimelic acids in the cell wall amino acid pattern of different species of a single genus, is not known. However, the latter seems to be the case, because the results demonstrate that morphologically very similar Actinoplanaceae sometimes have one or both of the diaminopimelic acids in their cells walls. The clearest example of this is found among the strains of Actinoplanes missouriensis (Table 3). Among these strains, which are morphologically very similar since they were placed by Couch (5) in the same species, are strains whose cell walls do not con-

9 VOL. 94, CELL WALL COMPOSITION IN ACTINOPLANACEAE 2045 tain DAPA and strains whose cell walls contain both DAPA and HDAPA. Although it is possible that the strains of A. missouriensis are a heterogeneous group representing more than one species, it is improbable that these morphologically similar strains represent species of different genera. Therefore, it seems justifiable to conclude that it is not unusual to find different diaminopimelic acids in the cell walls of species of a single genus, even though the type of diaminopimelic acid detected appears to be a reliable character at the species level. This conclusion is strengthened by the results obtained for the strains of Ampullariella lobata and A. campanulata (Table 5). These two species, which are by description morphologically very similar (5), are also chemically very similar. Galactosamine, the heptose, and, in most instances, the unidentified compound designated as the spot near xylose were unique to the cell walls of only these two species. However, the results again reveal that these morphologically and chemically very similar species do not have the same diaminopimelic acids in their cell walls. This again leads one to conclude that, while the amino acids in the cell walls of the Actinoplanaceae may be species-specific, they are probably not genus-specific, as was suggested for the grampositive bacteria studied by Cummins and Harris. Therefore, it does not seem wise at this time to propose for these Actinoplanaceae a classification in which each genus would include only species having the same cell wall amino acids. The results of the qualitative analyses also demonstrate that the species investigated have one of three basic patterns of cell wall monosaccharides. One of these monosaccharide patterns, as has already been noted, is characteristic of the cell walls of Ampullariella lobata and A. campanulata, as well as the two unidentified Ampullariella species, and provides evidence that they are closely related (Table 5). The detection of major amounts of the heptose, and minor amounts of galactosamine and the unknown sugar, diagnostically defines these two species in such a way that they are distinct among all the species examined. The other two basic patterns of cell wall monosaccharides are very similar, differing only by the presence or absence of the deoxyhexose which was tentatively identified as 6-deoxy-Lglucose. This monosaccharide characterizes the sugar patterns of all the strains of Actinoplanes philippinensis, A. utahensis, Amorphosporangium auranticolor, and Ampullariella regularis, and is absent from the sugar patterns of all but one of the strains of Pilimelia, Ampullariella digitata, and Actinoplanes missouriensis. The single strain which was the exception (A. missouriensis, strain 267, Table 3) seems very possibly misidentified, since it is chemically similar to most of the Actinoplanes utahensis strains (Table 3). As with the diaminopimelic acids in the amino acid patterns, the presence or absence of the deoxyhexose in the sugar patterns correlates well with the current species concepts, but not with current concepts of genera. Wbile this may be due simply to coincidence, it seems more probable that it again reflects the close relationship of all Actinoplanaceae presently placed in different genera. Evidence for this supposition is also furnished by the sugar analyses for the strains of Ampullariella regularis and Actinoplanes utahensis (Tables 2, 3). The strains included in these two species, besides having identical amino acids in their cell walls, exhibit very similar cell wall sugar patterns. In fact, one of the Ampullariella regularis strains (312) has a cell wall composition identical with one of the Actinoplanes utahensis strains (258). The finding of similar cell wall compositions among the strains of these two species supports the conclusion that they, if not all the strains exhibiting the deoxyhexose, are more closely related than is indicated by their generic positions. Further evidence for this conclusion is provided by the sugar analyses for Ampullariella digitata and three of the strains of Actinoplanes missouriensis (Tables 3, 4). Some of the strains included in both of these generically separated species have identical cell wall sugar and amino acid patterns. Although it is possible that the observed sugar patterns provide a basis for classifying species of Actinoplanaceae into genera, the quantitative amino acid and amino sugar analyses seem to dispel this idea (Table 6). These data further tend to substantiate that complex relationships exist among the Actinoplanaceae examined. In the previous discussion of the qualitative data, the Ampullariella lobata and A. campanulata strains (Table 5) appeared to stand apart from all the other Actinoplanaceae studied because of the detection of three unique cell wall sugars. The quantitative data, however, put these two species back into perspective in relation to the other Actinoplanaceae. Table 6 shows that Ampullariella campanulata and Amorphosporangium auranticolor have almost identical molar ratios of their cell wall amino acid components. In the same manner, Ampullariella lobata and the Ampullariella regularis strain 79 also have essentially identical molar ratios. These molar ratios are only slightly different from the molar ratios obtained for most of the remaining strains not having DAPA in their cell wall amino acid pat-

10 2046 SZANISZLO AND GOODER J. BACTERIOL. TABLE 6. Molar ratiosc of principal cell wall amino acids Organism (morphological designation) Amino acids Glutamic Glycine Alanine DAPA HDAPA Pilimelia aniulata P. terevasa Actinoplanes philippinensis A. missouriensis A. utahensis Ampullariella digitata A. lobata A. regularis strain A. regularis strain A. regularis strain A. campanulata Amorphosporangium auranticolor Average value of duplicate determinations (variation less than 3%). terns. Again, the data lead to the interesting paradox that species included in one genus of Actinoplanaceae are, in some respects, less similar to each other than they are to certain species of other genera of Actinoplanaceae. Both Yamaguchi (23) and Becker et al. (1) placed species ol Actinoplanes, Ampullariella, and Amorphosporangium in a single group on the basis of their cell wall constituents. However, these investigators did not study enough strains to speculate about whether the pattern of cell wall constituents could be used to group the Actinoplanaceae into genera and species. Becker et al. recorded only the constituents that he thought were present in the highest concentrations, and Yamaguchi recorded all the components he detected. More sugars were often detected in this study than were detected in the cell walls of the same strains by Yamaguchi or by Becker et al. The different observations probably resulted from chromatographing different equivalent amounts of cell walls. However, it is possible that some portion of the cell wall sugars may have been lost by Yamaguchi and Becker et al. during the cell disruptions by ultrasonic vibrations as compared to the mechanical disruption employed here. Similar effects, due to the method of cell disruption and the length of time disruption was conducted,havebeenreported previously (20,21). These cell wall data appear to lead to the conclusion that the present generic concepts for the Actinoplanaceae are inadequate and do not reflect the mixed patterns of chemical characters found among the species. The results favor placing the species examined in this work in a single genus. Results concerning the nature of pigment extracts of the intracellular carotenoids of these strains also strengthen the conclusion that these Actinoplanaceae are so closely related that they should be included in a single genus (22). This would reflect that, as presently known, these species have many characters in common, and differ from each other only by a limited number of features. Such a reorientation of these species into one genus would make the new genus analogous to the more thoroughly investigated genus Streptomyces which is known to exhibit extreme morphological variation among its species, particularly with regard to their sporogenous apparatus. Included in such a genus would be all the species examined, and which are now known to be not only morphologically distinct, but also chemically distinct. It is not suggested, however, that all the described genera of Actinoplanaceae should be included in a single genus. On the contrary, both Yamaguchi and Becker et al. presented evidence that the other genera of Actinoplanaceae, not examined in this work, exhibit unique variant conditions with regard to their cell wall amino acid patterns. These investigators found that Streptosporangium and Spirillospora were chemically distinct since glycine was not detected as a major component in their cell walls. They also determined that Microellobosporia was chemically more similar to Streptomyces than to the other Actinoplanaceae, because the cell walls of both these genera exhibited the LL-isomer of 2,6- DAPA. Based on their results, it appears that more strains of Streptosporangium, Spirillospora, and Microellobosporia will have to be studied before their relationship with the genera studied in this work can be adequately assessed. ACKNOWLEDGMENTS We are grateful to J. N. Couch for his encouragement and suggestions during the course of this work. We also thank Elizabeth Work, H. R. Perkins (Twy-

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