JOURNAL OF BACTERIOLOGY, Dec. 1967, p. 2037-2047 Vol. 94, No. 6 Copyright © 1967 American Society for Microbiology 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 27514

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, Amorpho- sporangium, 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 dis- tinct 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 substan- tiated that cross-generic similarities existed. Based on these results and the morpho- logical conclusions reached by other investigators, a single-genus concept is sug- gested for the Actinoplanaceae examined.

Existing schemes of bacterial classification often The present investigation was undertaken to need re-examination to determine whether tra- compare the cell wall composition of numerous ditional criteria are obscuring natural relation- strains of Actinoplanes, Ampullariella, and Amor- ships. Among the Actinoplanaceae (Actinomy- phosporangium to determine whether the tra- cetales), many species are placed in their genus ditional criteria are obscuring natural relation- category on the basis of a relatively few morpho- ships among species presently classified in these logical characters. This often necessitates classi- genera. Three strains of keratinophilic Actino- fying similar Actinoplanaceae into different planaceae were also examined. Couch (5) specu- genera. The most notable examples occur among lated that such Actinoplanaceae probably belong the species which are morphologically and cul- in the genus Ampullariella. However, Kane (12) turally very similar to Actinoplanes philippinensis, reported that morphological and cultural differ- the species on which the family Actinoplanaceae ences warranted placing the keratinophilic strains is based. These species are classified among three she studied in a new genus which she named genera predominantly on the basis of sporangio- Pilimelia. spore morphology (5, 6). Species producing While this investigation was in progress, sporangiospores morphologically similar to those Yamaguchi (23) and Becker, Lechevalier, and produced by Actinoplanes philippinensis (sub- Lechevalier (1) reported on the cell wall compo- spherical with a tuft of are sition of various aerobic actinomycetes. Their polar flagella) placed investigations revealed that strains of Actino- in the genus Actinoplanes. Species producing rod- planes, Ampullariella, and Amorphosporangium shaped sporangiospores with a tuft of polar flag- had similar, but not necessarily identical, cell ella are placed in the genus Ampullariella, and wall compositions. However, the investigations species producing rod-shaped sporangiospores of Yamaguchi and Becker et al. did not involve with no flagella are placed in the genus Amor- sufficient numbers of these Actinoplanaceae to phosporangium. allow for extensive speculation on the validity of 1 Part of a dissertation submitted to the faculty of their classification among three genera. the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree MATERIALS AND METHODS of Doctor of Philosophy in the Department of Botany. Organisms. The name, number, and general loca- 2 Present address: Laboratory of Applied Micro- tion of the original collection site of each laboratory biology, Division of Engineering and Applied Physics, stock strain examined are listed in Table 1. The strains Harvard University, Cambridge, Mass. 02138. of Actinoplanes, Ampullariella, and Amorphosporan- 2037 2038 SZANISZLO AND GOODER J. BACTERIOL. TABLE 1. Name, number, and general location of original collection site of each strain investigated

Organism Strain no. Collection site I ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~I_ Actinoplanes philippinensis...... 2 Philippine Islands Actinoplanes utahensis...... 258 Salt Lake City, Utah 259 Salt Lake City, Utah 260 Salt Lake City, Utah 261 Dunphy, Nev. Actinoplanes missouriensis...... 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. Ampullariella regularis...... 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 Ampullariella digitata...... 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. Ampullariella lobata...... 72 Madison, Wis. 74 Madison, Wis. 337 Draper, Va. Ampullariella campanulata...... 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 Ampullariella sp...... 1492 Bombay, India 1539 Mahabalipuram, India Amorphosporangium auranticolor. 253 Dunphy, Nev. 262 Dunphy, Nev. Pilimelia anulata...... 1 Walkerton, Ind. Pilimelia terevasa...... 1 Walkerton, Ind. Pilimelia sp...... 1777 Hyderabad, India gium were identified by J. N. Couch (3, 4, 5, 6), and 1,000 ml of Czapek Dox broth (Difco), fortified with stock cultures of each strain are maintained in his 5 g of peptone (Difco), housed in 1,500-ml low-form laboratory on Czapek agar (Difco) and peptone- flasks, by suspending adequate amounts of each Czapek agar (Czapek agar fortified with 5 g/l ,000 ml strain in 50 ml of sterile broth and blending for 60 of Difco peptone). The strains of Pilimelia were sec at high speed in a cold Waring Blendor micro-cup. identified by W. D. Kane (12) and are maintained After inoculation, the cultures were grown for 5 days by her on peptone-Czapek agar. at 24 to 26 C with continuous agitation (100 strokes/ Medium and culture method. The organisms from min) on a gyratory shaker (model G-10, New Bruns- 3-week-old slants were prepared for inoculation into wick Scientific Co., New Brunswick, N.J.). The re- VOL. 94, 1967 CELL WALL COMPOSITION IN ACTINOPLANACEAE 2039 sulting vegetative hyphae were harvested by centrifu- both separately and after admixture with the hy- gation at 3,000 X g and washed three times with drolytic samples. distilled water. Galactose and glucose identification. When both Preparation of cell walls. The hyphae were me- galactose and glucose were present together, as in the chanically disrupted in a Braun homogenizer by the hydrolysate of walls of Actinoplanes philippinensis method of Bleiweis, Karakawa, and Krause (2). strain 2, the identity of each was verified by adding Disrupted suspensions were centrifuged at 10,000 X g separately and together glucose oxidase (0.5 unit) for 10 min. Hyphae with any remaining intact cells and galactose oxidase (0.125 unit) to hydrolysate in their length formed a tightly packed, colored layer samples containing 0.8-mg equivalent of wall material. at the bottom of the centrifuge tube. Completely Enzyme mixtures were incubated for 2 hr at 37 C, disrupted hyphae were devoid of color and formed a and the entire sample was then chromatographed to loosely packed, upper white layer, which was rinsed away from the colored layer by repeated washes with test for the disappearance of the respective sugar or distilled water. The cell walls were collected by sugars. centrifugation, treated for two periods of 1 hr each Deoxyhexose identification. Relatively pure samples in 1% sodium lauryl sulfate, rewashed thoroughly in of the fast-running sugar detected in wall hydrolysates distilled water, and lyophilized. were obtained by chromatographing as a band the Enzyme treatment of walls. Lyophilized walls in combined H2SO4 hydrolysates of 37 mg of cell walls, 100-mg amounts were treated with 10 mg of crystalline with BAW as solvent. After the position of the sugar ribonuclease in 10 ml of 0.1 M phosphate buffer was located with test strips, the band was eluted from (pH 7.8) for 3 hr at 37 C, then 5 mg of crystalline the paper and the eluate filtered through a washed trypsin was added, and incubation was continued filter, 0.45 , pore size (Millipore Corp., Bedford, for 3 hr at 37 C. The walls were deposited by centrifu- Mass.), and then taken to dryness over P205. Wash gation, washed three times with distilled water, sus- water from a blank chromatogram was similarly pended in 20 ml of 0.2 N HCI containing crystalline treated and used as a control. Both residues were pepsin (1 mg/ml), and incubated overnight at 37 C. taken up in 2 ml of distilled water. Samples of the The walls were collected by centrifugation, thoroughly sugar solution and control were subjected to the washed with distilled water, lyophilized, and used in Dische spectrophotometric procedure (9) for 6-deoxy- all subsequent analyses. hexoses. Various concentrations of similar samples Preparation of samples for chromatography. Paper were also chromatographed overnight with rhamnose chromatographic analysis of the walls of each strain and fucose, separately and together, with n-butanol, for amino acids and amino sugars was conducted pyridine, and water (6:4:3, v/v) as the solvent (13), after hydrolysis of 10-mg samples of lyophilized in order to determine the R rhamnose (R,h) of the un- walls in 5 ml of 6 N HCI in sealed Pyrex tubes for 16 known. hr at 100 C. For cell wall sugars, the hydrolysis Diaminopimelic acid identifications. The paper conditions were 20 mg of lyophilized walls in 2 ml of chromatographic separation of 2, 5-diamino-3-hy- 2 N H2SO4 in sealed Pyrex tubes for 2 hr at 100 C. droxypimelic acid (HDAPA) and the stereoisomers Subsequent handling of the hydrolysates was sub- of 2, 6-diaminopimelic acid (DAPA) was accom- stantially as described by Michel and Gooder (15). plished with the general method of Hoare and Work Residues from the HCI hydrolysates and H2SO4 (10), but with the solvent suggested by Perkins (18). hydrolysates were dissolved in 0.4 ml and 0.25 ml The equivalent of 0.5 mg of cell walls of each strain of distilled water, respectively. Cell wall samples in 5- was chromatographed. mg amounts were prepared for quantitative chroma- Quantitative amino acid analysis. Quantitative tography as described by Moore and Stein (16). chromatography was conducted using a Beckman Paper chromatography. Two-dimensional descend- model 120 amino acid analyzer. The equivalent of 1 was conducted on What- mg of HCl-hydrolyzed cell walls was examined, and ing paper chromatography duplicate determinations were made for the acid and man no. 1 filter paper, with n-butanol, acetic acid, neutral amino acids; however, since major amounts and water (BAW; 60:10:20, v/v) as the first solvent of the basic amino acids were not detected, only single and 2,6-lutidine (Practical, Matheson, Coleman, and runs were made for their quantification. Bell, Cincinnati, Ohio) and water (LW; 65:35, v/v) Electron microscopy. Electron microscope prepa- as the second solvent. For amino acids and amino rations were made by the method of Sharp (19). sugars, the hydrolytic equivalent of 0.5 or 1.0 mg of cell walls was applied to the paper, which was subse- RESULTS quently developed for 23 hr with BAW and 28 hr with LW. Spots were revealed with ninhydrin. For sugars, Criteria Of purity. The criteria for purity of all the hydrolytic equivalent of 0.8 mg and 1.6 mg of wall fractions were: absence of color in disrupted walls was applied to papers which were developed hyphae packed by centrifugation, and absence of for 30 hr with BAW and 20 hr with LW. Sugar spots large amounts of particulate matter inside the were revealed with acid aniline phthalate reagent. disrupted cells when examined with the electron All samples were chromatographed at least two times microscope. Wall fractions obtained from the and verification of the components was accomplished, initial disruption of hyphae, when examined with where possible, by using pure substances as markers the electron microscope, rarely exhibited hyphae subjected to identical chromatographic conditions with intact cells. Small amounts of particulate 2040 SZANISZLO AND GOODER J. BACTERIOL. matter were noticed along the lengths of some Analyses of the cell walls of the strains of hyphae (Fig. 1). Examination of the enzyme- Actinoplanes utahensis and A. missouriensis re- treated cell walls showed that most ofthis material vealed somewhat different patterns (Table 3). was removed during the enzymatic digestions The distinguishing cell wall components for A. (Fig. 2). utahensis are HDAPA, galactose, and mannose, Qualitative cell wlall analyses. Cell wall prepa- and those for A. missouriensis are galactose, glu- rations were made from 48 strains of Actino- cose, and either HDAPA or both HDAPA and planaceae previously assigned to 10 species and DAPA. Various other components were detected the four genera Actinoplanes, Ampullariella, in low concentrations. The strains of A. utahensis Amorphosporangium, and Pilimelia. Hydrolytic usually showed traces of xylose and the 6-deox- residues of these cell walls, when subjected to yhexose. In contrast, the strains of A. missouri- paper chromatography, revealed the substances ensis showed traces of mannose, arabinose, and documented in Table 2-5. The sugars and DAP xylose. Strain 267 had a cell wall composition acids are expressed in relative amounts according more like A. utahensis than A. missouriensis. to the sizes and intensities of their spots on paper. Results for the strains of A. missouriensis indi- The relative amounts of glucosamine, muramic cated that these strains are in reality a hetero- acid, glycine, and alanine are not shown because geneous group composed of two chemically dis- all of these compounds were present in large tinct subgroups. Cell walls of strain 260 were re- amounts (4 + or greater) in each hydrolytic ported by Yamaguchi to contain galactose, sample. The grouping of the organisms is pre- arabinose, and major amounts of both HDAPA sented so as to facilitate discussion, and does not and DAPA. imply relatedness. Table 4 lists results for strains of Pilimelia and Strains of Actinoplanes philippinensis, Amor- Ampullariella digitata. The three strains of phosporangium auranticolor, and Ampullariella Pilimelia were distinguished by having cell walls regularis are listed in Table 2. All strains of these containing DAPA, galactose, glucose, mannose, morphologically determined species, except strain arabinose, and xylose. Those of Ampullariella 28 and strain 312, exhibited nearly identical cell digitata had cell walls containing HDAPA and wall sugar patterns. Actinoplanes philippinensis all or some of the same sugars. These latter was distinguished from the other two species by strains showed more variation than was found in not having HDAPA in its cell walls. In contrast, any other morphologically determined group. the strains of Amorphosporangium auranticolor Strain 33 was also studied by Yamaguchi. He were distinguished by having both HDAPA and similarly reported only galactose and the pink DAPA in their cell walls, and the strains of Am- spot (xylose) in this strain. The strains of Pili- pullariella regularis were distinguished by not melia are interesting because they were the only having DAPA in their cell walls. Of the two strains strains that showed minor amounts of galactose. of A. regularis (strains 28 and 312) that differed In addition, they were the only strains, besides in their general cell wall sugar constituents, only Actinoplanes philippinensis strain 2, which ex- strain 28 differed extensively, showing simply hibited major amounts of DAPA in the absence xylose and the deoxyhexose. The absence of galac- of HDAPA. Chemically, the Pilimelia sp. seemed tose in the cell walls of this organism made it more like P. anulata than P. terivasa. unique among the 48 strains investigated. The results obtained for Ampullariella lobata Yamaguchi (23) analyzed the cell walls of three and Ampullariella campanulata revealed that of the strains listed in Table 2, while Becker et these organisms are easily distinguished from all al. (1) analyzed one. Actinoplanes philippinensis the others (Table 5). Only the cell walls of these strain 2 was reported by Yamaguchi to have the strains contained the heptose, small amounts of cell wall sugars galactose, arabinose, and man- galactosamine, and in some instances an un- nose, as well as two sugars he was not able to known (described simply as the spot near xylose). identify. These two unidentified sugars are prob- In addition, cell walls of all the strains contained ably the sugars identified as xylose and 6-de- galactose and mannose in large amounts. Trace oxyhexose in this work. Amorphosporangium amounts of other components were sometimes auranticolor strain 253 was examined by both detected. Two of the three strains designated Yamaguchi and Becker et al. Yamaguchi did not Ampullariella lobata could be distinguished from detect xylose or the 6-deoxyhexose in the walls of the remaining strains by the absence of DAPA. this strain, but Becker et al. reported that this The unidentified strains of Ampullariella seemed strain had no sugar in major amounts. The third clearly to belong in this Ampullariella lobata and strain examined by Yamaguchi was Ampullar- A. campanulata complex of organisms. iella regularis strain 28. He reported that this Deoxyhexose. Deoxyhexose, first detected in the strain had no sugars. cell walls of Actinoplanes philippinensis, gave a 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 sul- fate, enzyme-treated with ribonuclease, pepsin, and trypsin, and then shadowed with platinum-palladium. Note absence ofparticulate matter. X 25,000. 2042 SZANISZLO AND GOODER J. BACTERIOL. brown color when sprayed with acid aniline the Rrh previously reported for fucose (13), in- phthalate reagent and was thought to be rham- dicating that the different values could be accu- nose. However, when authentic rhamnose was rately compared. These results indicate that the added to the sugar residues before chromatog- sugar is 6-deoxy-L-glucose; however, sufficient raphy, the resulting sugar spot became abnor- quantities of the sugar are as yet unavailable for mally elongated. Reaction of the isolated a more definite identification. unknown sugar with cysteine-sulfuric acid (9) Galactose and glucose. Cell wall hydrolytic revealed that the chromophore formed resembled residues from Actinoplanes philippinensis had two that formed by rhamnose and fucose with an components corresponding to glucose and galac- absorption maximum at 400 m,u. All of the six tose. Residues treated withglucose oxidase showed other 6-deoxyhexoses are reported to resemble a single hexose spot corresponding to galactose rhamnose and fucose in this test (14). after chromatography, and residues treated with Paper chromatography of the unknown sugar, galactose oxidase showed a single hexose spot fucose, and rhamnose showed that the (Rrh) of corresponding to glucose after chromatography. the unknown (0.96 to 0.97) compared favorably Residues treated with both enzymes showed no with the Rrh reported for 6-deoxy-L-glucose (13). spots corresponding to either glucose or galactose The Rrh for fucose also compared favorably with after chromatography.

TABLE 2. Components in cell walls of Actinoplanes philippinensis, Amorphosporangium auranticolor, and Ampullariella regularisa

Galac- Man- Arabi- Spot Deoxy- Organism Strain DAPA IHDAPA tos- Heptose Galac- Glucose Xylose near hexose (morphological designation) no. amine tose nose nose xylose A. philippinensis..... 2 +++ ++ ++ ++ ++ ++ A. auranticolor...... 253 ++ ++ ++ ++ ++ ++ 262 ++ ++ ++ ++ ++ ++ A. regularis...... 79 ++ ++ ++ ++ ++ ++ 154 ++ ++ ++ ++ ++ 164 ++ ++ ++ ++ ++ 168 ++ ++ ++ ++ ++ 395 ++ ++ ++ ++ ++ 850 ++ ++ ++ ++ ++ 915 ++ ++ ++ ++ 312 Tr + ±+ ++ 28

- All contain major amounts of glucosamine, muramic acid, glutamic acid, glycine, and alanine. Components graded: +++, ++, +, Tr (Trace).

TABLE 3. Components in cell walls of Actinoplanes utahensis and A. missouriensisa

Strain Galac- Arabi- Spot Deoxy- Organism DAPA HDAPA tos- Heptose Galac- Glucose Man- Xylose near hexose (morphological designation) no. amine itose Inose Inose xylose

A. utahensis ...... 258 ++ Tr Tr Tr 259 Tr ++ Tr Tr 260 ++ Tr Tr 261 ++ Tr A. missouriensis...... 267 ++ + Tr Tr 431 ++ ++ Tr Tr Tr 443 ++ ++ Tr Tr 825 Tr Tr Tr 221 ++ + Tr Tr Tr Tr Tr Tr 222 ++ + ++ 263 ++ ++ Tr Tr Tr 657 ++ ++ Tr Tr Tr

a All contain major amounts of glucosamine, muramic acid, glutamic acid, glycine, and alanine. Components graded: +++, ++, +, Tr (Trace). 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 Deoxy- designation) no. amine ns noexylose hxs

P. terivasa ..... I +++ + +++ +++ ++ + P. anulata..... I +++ + +++ Tr ++ ++ Pilimelia sp.... 1 353 +++ + +++ Tr ++ ++ A. digitata..... 71 +++ +++ ++ + + + 118 ++± ++ Tr ++ ++ 131 +++ +++ ++ + + + .399 + + + + 370 +++ +++ + + + 386 + + +++ Tr + + 137 +++ +++ + + 33 +++ +++ ++ a All contain major amounts of glucosamine, muramic acid, glutamic acid, glycine, and alanine. Components graded: +++, ++, +, Tr (Trace).

TABLE 5. Components in cell walls of Ampullariella lobata and A. campaniulata-

Organism Galac- Spot (morphological Strain DAPA HDAPA tos- Heptose Galac- Glucose Man- Arabi- Xylose near Deoxy- designation) no. amine tose nose nose xylose hexose

A. lobata...... 72 Tr ++ ++ Tr Tr 74 Tr ++ Tr Tr Tr 337 ++ ++ Tr ++ ++ Tr Tr Tr A. campaniulata.. 65 ++ + Tr ++ ++ 126 ++ + Tr ++ Tr ++ 151 ++ Tr ++ ++ 182 ++ + Tr ++ + ++ Tr Tr 640 ++ Tr ++ ++ Tr Tr Tr ++ ++ 641 ++ Tr ++ Tr Tr Tr 642 ++ + Tr ++ Tr ++ Tr 643 ++ Tr ++ Tr ++ Tr Ampulariella sp.. 1492 ++ + Tr ++ + ++ Tr Tr Tr 1539 ++ Tr ++ ++ ++ Tr Tr Tr a All contain major amounts of glucosamine, muramic acid, glutamic acid, glycine, and alanine. Components-graded: +++, ++, +, Tr (Trace). Heptose and the compound with a mobility slowly than galactose and produce a violet-green similar to xylose. The heptose and the compound coloration with the p-anisidine hydrochloride that had a mobility similar to xylose posed special reagent. On the basis of thesefacts, this compound problems in identification. Both gave hexose was tentatively identified as a heptose sugar. (brown) colors with acid aniline phthalate rea- The compound that had a mobility similar to gent, but did not exhibit RF values similar to the xylose gave a brownish color with both acid more common hexose sugars. The heptose mi- aniline phthalate reagent and p-anisidine hy- grated more slowly than galactose and was at drochloride, and is possibly a 2-deoxy sugar or a first thought to be a uronic acid. However, when methylated aldohexose. However, many classes co-chromatographed with authentic glucuronic of compounds have constituents exhibiting mo- and galacturonic acid, the compound migrated bilities similar to this compound. more slowly and did not produce the red color Isomers of 2,6-DAPA. Paper chromatographic reported to be produced with p-anisidine phos- separation of the isomers of the 2,6-DAP acids phate or p-anisidine hydrochloride (11, 17). The showed that meso-DAPA or DD-DAPA, or color produced with the p-anisidine phosphate both, occurred in high concentrations and were was yellow-brown; the color with p-anisidine hy- the main isomers being detected. It is interesting drochloride was violet-brown. Davies (8) re- to note that traces of LL-DAPA were always de- ported that some aldoheptoses migrate more tected whenever meso- or DD-DAPA, or both, 2044 SZANISZLO AND GOODER J. BACTERIOL. were detected in major amounts. Also, traces of media. The results of the present investigation meso- or DD-DAPA, or both, were always de- are in accord with the current morphological tected when HDAPA occurred alone. Whenever groupings of the Actinoplanaceae at the species HDAPA was detected, it was found to occur in level. However, the same results indicate that high concentrations. Yamaguchi (23) also re- these morphological characters may not be ade- ported the occurrence of small amounts of the quate, when considered alone, for the classifi- 2,6-DAPA acids in the cell walls of the five pre- cation of Actinoplanaceae at higher levels. This viously mentioned organisms that he studied. conclusion is reached by comparing results from It seems unlikely that these trace constituents the qualitative analyses of the sugars, amino sug- are integral components of the cell wall; they ars, and amino acids found in the vegetative cell are, in all probability, of cytoplasmic origin. walls and the quantitative determinations of the Quantitative cell wall analyses. Results for the vegetative cell wall amino acids and amino sug- amino acid determinations of the walls of the ars. When these features are considered in com- type organisms are presented in Table 6. The bination with the conclusions of Couch (3, 4, values obtained for glucosamine and muramic 5, 6) and Kane (12), patterns are discerned which acid are not included, because they showed more suggest that the species examined are more closely than the 3% maximal variation obtained for related to one another than their present generic the duplicate amino acid determinations; they positions would indicate. seemed always to represent a 1 to 1 relationship. Qualitative analyses of the vegetative cell walls Amino acid molar ratios were calculated with show that every strain examined has basically glutamic acid as unity. Results indicate that all the same pattern of amino acid and amino sugar the type organisms have very similar molar ratios constituents. As was also reported by Yamaguchi for the amino acids in their cell walls. Glycine is (23), major amounts of glucosamine, muramic usually present in the largest amount. The acid, glycine, alanine, glutamic acid, and either amino acids in the cell walls of the two Pili- meso- or DD-DAPA (or both) and HDAPA (or melia strains appeared to be present in a 1:1:1:1 both) are found in the cell walls of Actinoplanes, relationship, and those in Actinoplanes philip- Ampullariella, and Amorphosporangium. The pinensis appeared to represent a 2:2:1:2 relation- present work adds Pilimelia to this list. ship, with alanine being present in the smallest In contrast to the statement of Cummins and amount. Those strains which did not have DAPA Harris (7), that each genus of gram-positive bac- all had somewhat different molar ratios, but teria appears to have a characteristic pattern of such variation could be explained, in most in- amino acids associated with its cell walls, two stances, as resulting from small amounts of con- genera of Actinoplanaeceae examined in this work taminating amino acids of cytoplasmic origin. have slightly different amino acid patterns among The Ampullariella campanulata and Amorpho- their species. It was observed that, in the cell walls sporangium auranticolor strains possessed signif- of species of these genera, some species contain icant amounts of cell wall HDAPA and DAPA. DAPA, some species HDAPA, and some species The sum total of the two DAP's was only slightly contain both these diaminopimelic acids in addi- different from the values obtained for the two tion to the glutamic acid, glycine, and alanine DAP's when they occurred alone. The Ampul- amino acid complement which was present in the lariella regularis strain 28 was also studied quan- cell walls of every strain examined. Whether this titatively by Perkins (18). Recalculations of his variation indicates that the Actinoplanaceae results showed that he obtained similar values for strains previously placed in the same genus but this organism, even though it was grown and in different species are in reality unrelated, or treated in a different way. Thus, all strains of the whether it means that it is not unusual to find Actinoplanaceae so far examined contain less various diaminopimelic acids in the cell wall alanine than glutamic acid in their cell walls. amino acid pattern of different species of a single genus, is not known. However, the latter seems DISCUSSION to be the case, because the results demonstrate The taxonomy of the Actinoplanaceae is based that morphologically very similar Actinoplana- predominantly on morphological characters and ceae sometimes have one or both of the diamino- a limited number of physiological features. The pimelic acids in their cells walls. The clearest ex- characters most commonly cited as being impor- ample of this is found among the strains of Ac- tant taxonomically are sporangiospore and tinoplanes missouriensis (Table 3). Among these sporangium shape, sporangiospore flagellation strains, which are morphologically very similar pattern, mode of branching or coiling of the since they were placed by Couch (5) in the same sporogenic hypha, and coloration of cultural species, are strains whose cell walls do not con- VOL. 94, 1967 CELL WALL COMPOSITION IN ACTINOPLANACEAE 2045 tain DAPA and strains whose cell walls contain and Actinoplanes missouriensis. The single strain both DAPA and HDAPA. Although it is possible which was the exception (A. missouriensis, strain that the strains of A. missouriensis are a hetero- 267, Table 3) seems very possibly misidentified, geneous group representing more than one species, since it is chemically similar to most of the Actino- it is improbable that these morphologically planes utahensis strains (Table 3). As with the similar strains represent species of different diaminopimelic acids in the amino acid patterns, genera. Therefore, it seems justifiable to conclude the presence or absence of the deoxyhexose in that it is not unusual to find different diamino- the sugar patterns correlates well with the current pimelic acids in the cell walls of species of a single species concepts, but not with current concepts genus, even though the type of diaminopimelic of genera. Wbile this may be due simply to coin- acid detected appears to be a reliable character cidence, it seems more probable that it again re- at the species level. flects the close relationship of all Actinoplanaceae This conclusion is strengthened by the results presently placed in different genera. Evidence for obtained for the strains of Ampullariella lobata this supposition is also furnished by the sugar and A. campanulata (Table 5). These two species, analyses for the strains of Ampullariella regularis which are by description morphologically very and Actinoplanes utahensis (Tables 2, 3). The similar (5), are also chemically very similar. strains included in these two species, besides hav- Galactosamine, the heptose, and, in most in- ing identical amino acids in their cell walls, ex- stances, the unidentified compound designated hibit very similar cell wall sugar patterns. In fact, as the spot near xylose were unique to the cell one of the Ampullariella regularis strains (312) walls of only these two species. However, the has a cell wall composition identical with one of results again reveal that these morphologically the Actinoplanes utahensis strains (258). The find- and chemically very similar species do not have ing of similar cell wall compositions among the the same diaminopimelic acids in their cell walls. strains of these two species supports the conclu- This again leads one to conclude that, while the sion that they, if not all the strains exhibiting the amino acids in the cell walls of the Actinoplana- deoxyhexose, are more closely related than is in- ceae may be species-specific, they are probably dicated by their generic positions. Further evi- not genus-specific, as was suggested for the gram- dence for this conclusion is provided by the sugar positive bacteria studied by Cummins and Harris. analyses for Ampullariella digitata and three of Therefore, it does not seem wise at this time to the strains of Actinoplanes missouriensis (Tables propose for these Actinoplanaceae a classification 3, 4). Some of the strains included in both of in which each genus would include only species these generically separated species have identical having the same cell wall amino acids. cell wall sugar and amino acid patterns. The results of the qualitative analyses also Although it is possible that the observed sugar demonstrate that the species investigated have patterns provide a basis for classifying species of one of three basic patterns of cell wall mono- Actinoplanaceae into genera, the quantitative saccharides. One of these monosaccharide pat- amino acid and amino sugar analyses seem to terns, as has already been noted, is characteristic dispel this idea (Table 6). These data further tend of the cell walls of Ampullariella lobata and A. to substantiate that complex relationships exist campanulata, as well as the two unidentified among the Actinoplanaceae examined. In the pre- Ampullariella species, and provides evidence that vious discussion of the qualitative data, the they are closely related (Table 5). The detection Ampullariella lobata and A. campanulata strains of major amounts of the heptose, and minor (Table 5) appeared to stand apart from all the amounts of galactosamine and the unknown other Actinoplanaceae studied because of the de- sugar, diagnostically defines these two species in tection of three unique cell wall sugars. The such a way that they are distinct among all the quantitative data, however, put these two species species examined. back into perspective in relation to the other The other two basic patterns of cell wall Actinoplanaceae. Table 6 shows that Ampul- monosaccharides are very similar, differing only lariella campanulata and Amorphosporangium by the presence or absence of the deoxyhexose auranticolor have almost identical molar ratios of which was tentatively identified as 6-deoxy-L- their cell wall amino acid components. In the glucose. This monosaccharide characterizes the same manner, Ampullariella lobata and the Am- sugar patterns of all the strains of Actinoplanes pullariella regularis strain 79 also have essentially philippinensis, A. utahensis, Amorphosporangium identical molar ratios. These molar ratios are auranticolor, and Ampullariella regularis, and is only slightly different from the molar ratios ob- absent from the sugar patterns of all but one of tained for most of the remaining strains not the strains of Pilimelia, Ampullariella digitata, having DAPA in their cell wall amino acid pat- 2046 SZANISZLO AND GOODER J. BACTERIOL. TABLE 6. Molar ratiosc ofprincipal cell wall amino acids

Amino acids Organism (morphological designation) Glutamic Glycine Alanine DAPA HDAPA

Pilimelia aniulata ...... 1.00 1.16 1.03 0.91 P. terevasa ...... 1.00 1.08 0.83 1.11 Actinoplanesphilippinensis . . 1.00 1.08 0.57 1.06 A. missouriensis ...... 1.00 1.13 0.80 0.70 A. utahensis...... 1.00 1.18 0.93 0.53 Ampullariella digitata ...... 1.00 1.19 0.73 0.64 A. lobata ...... 1.00 1.24 0.78 0.75 A. regularis strain 28 ...... 1.00 1.21 0.67 0.87 A. regularis strain 79 ...... 1.00 1.23 0.78 0.77 A. regularis strain 168 ...... 1.00 1.41 0.86 0.66 A. campanulata...... 1.00 1.15 0.69 0.52 0.38 Amorphosporangium auranticolor ...... 1.00 1.14 0.72 0.53 0.31 Average value of duplicate determinations (variation less than 3%). terns. Again, the data lead to the interesting be included in a single genus (22). This would paradox that species included in one genus of reflect that, as presently known, these species Actinoplanaceae are, in some respects, less similar have many characters in common, and differ from to each other than they are to certain species of each other only by a limited number of features. other genera of Actinoplanaceae. Such a reorientation of these species into one Both Yamaguchi (23) and Becker et al. (1) genus would make the new genus analogous to placed species ol Actinoplanes, Ampullariella, and the more thoroughly investigated genus Strepto- Amorphosporangium in a single group on the myces which is known to exhibit extreme mor- basis of their cell wall constituents. However, phological variation amongits species, particularly these investigators did not study enough strains to with regard to their sporogenous apparatus. In- speculate about whether the pattern of cell wall cluded in such a genus would be all the species constituents could be used to group the Actino- examined, and which are now known to be not planaceae into genera and species. Becker et al. only morphologically distinct, but also chemically recorded only the constituents that he thought distinct. were present in the highest concentrations, and It is not suggested, however, that all the de- Yamaguchi recorded all the components he de- scribed genera of Actinoplanaceae should be in- tected. More sugars were often detected in this cluded in a single genus. On the contrary, both study than were detected in the cell walls of the Yamaguchi and Becker et al. presented evidence same strains by Yamaguchi or by Becker et al. that the other genera of Actinoplanaceae, not The different observations probably resulted from examined in this work, exhibit uniquevariant con- chromatographing different equivalent amounts ditions with regard to their cell wall amino acid of cell walls. However, it is possible that some patterns. These investigators found that Strepto- portion of the cell wall sugars may have been lost sporangium and Spirillospora were chemically by Yamaguchi and Becker et al. during the cell distinct since glycine was not detected as a major disruptions by ultrasonic vibrations as compared component in their cell walls. They also deter- to the mechanical disruption employed here. mined that Microellobosporia was chemically Similar effects, due to the method of cell dis- more similar to Streptomyces than to the other ruption and the length of time disruption was Actinoplanaceae, because the cell walls of both conducted,havebeenreported previously (20,21). these genera exhibited the LL-isomer of 2,6- These cell wall data appear to lead to the con- DAPA. Based on their results, it appears that more clusion that the present generic concepts for the strains of Streptosporangium, Spirillospora, and Actinoplanaceae are inadequate and do not reflect Microellobosporia will have to be studied before the mixed patterns of chemical characters found their relationship with the genera studied in this among the species. The results favor placing the work can be adequately assessed. species examined in this work in a single genus. Results concerning the nature of pigment extracts ACKNOWLEDGMENTS of the intracellular carotenoids of these strains We are grateful to J. N. Couch for his encourage- also strengthen the conclusion that these Actino- ment and suggestions during the course of this work. planaceae are so closely related that they should We also thank Elizabeth Work, H. R. Perkins (Twy- VOL. 94, 1967 CELL WALL COMPOSITION IN ACTINOPLANACEAE 2047 ford Laboratories, Ltd., London), and J. M. Stewart methylated derivatives on the paper chroma- (Rockefeller University) for providing authentic togram. J. Chem. Soc., p. 1702-1706. samples of the various diaminopimelic acids. 12. KAN, W. D. 1966. A new genus of the Actino- This investigation was supported by a grant to planaceae, Pilimelia, with a description of two J. N. Couch from the Eli Lilly Company, and by species, Pilimelia terevasa and Pilimelia anulata. Public Health Service grant Al 07381 from the J. Elisha Mitchell Sci. Soc. 82:220-230. 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