Kibdelosporangium Philippinense Sp. Nov. Isolated from Soil

INTERNATIONAL JOURNAL OF SYSTEMATICBACTERIOLOGY, July 1988, p. 282-286 Vol. 38, No. 3 0020-7713/88/030282-05$02.OO/O Copyright 0 1988, International Union of Microbiological Societies

Kibdelosporangium philippinense sp. nov. Isolated from Soil

FREDERICK P. MERTZ* AND RAYMOND C. YAO The Lilly Research Laboratories, Eli Lilly & Company, Indianapolis, Indiana 46285

A new species of Kibdelosporungium is described. This organism is characterized by white aerial hyphae bearing long chains of smooth-walled spores and abundant sporangiumlike structures. The new species contains meso-diaminopimelic acid, arabinose, and galactose (type IV cell walls, type A whole-cell sugar pattern), phosphatidylethanolamine (type PI1 phospholipid pattern), and no mycolic acids. The single soil isolate has chemical and morphological properties consistent with the genus Kibdelosporungium. A comparison with previously described species of the genus Kibdelosporungium in which we used standard techniques, fatty acid profiles, and polypeptide banding fingerprints indicated that this strain is a new species. The name proposed for this new species is Kibdelosporungium philippinense. The type strain is strain A80407 (= NRRL 18198).

Taxonomy of the Actinomycetales is constantly changing Media. Cells were grown for 72 h at 30°C in medium iis new genera are discovered and as workers develop new containing 30 g of tryptic soy broth, 3 g of yeast extract, 2 g techniques which challenge existing classifications. Isolates of MgSO, . 7H,O, 5 g of glucose, and 4 g of maltose in 1,000 ?with type IV cell walls (10) (i.e., meso-diaminopimelic acid ml of deionized water. After incubation on a rotary shaker, plus arabinose and galactose in whole-cell hydrolysates) and the cells were harvested by centrifugation, washed twice llacking mycolic acids have recently undergone a major with sterile water, and then used as inocula. Cultural studies rearrangement. Two new nocardioform genera, Amycolata were done on ISP media 2 to 9 (16), calcium malate agar (19), and Amycolatopsis, were erected by Lechevalier et al. (11) Czapek solution agar (19), glucose-yeast extract agar (4), ’to accommodate mycolateless organisms formerly classified nutrient agar (4), tomato paste-oatmeal agar (19), and tap in the genus Nocardia. Another new genus, Saccharothrix water agar (4). ((71,although not characterized by type IV cell walls, has also Cultural observations. Characteristics were recorded after ,absorbed a species formerly included among the nocardiae 21 days of incubation at by using methods recom- ((6).The genus Kibdelosporangium, recently described by 30°C Shearer et al. (14), also has type IV cell walls lacking mended by Shirling and Gottlieb (16). The ISCC-NBS Cen- mycolic acids, and its members produce both long chains of troid Color Charts (18) were used to identify reverse side aerial spores and sporangiumlike structures. Development colors; color chips from the Color Harmony Manual (3)were or release of spores from these structures has not been used to describe aerial mycelia. ldemonstrated. Physiological tests. Methods recommended by Gordon et Kibdelosporangia are known to produce novel glycopep- al. (4) were used for the physiological tests. H,S production tide antibiotics like the ardacins. Although it has been was measured by placing lead acetate strips (Difco Labora- reported (14) that 10 isolates have been collected, only 2 tories, Detroit, Mich.) over the surfaces of ISP medium 6 have been described previously (14, 15). agar slants. Testosterone degradation was measured by In the course of isolating microorganisms for our antibiot- using 0.1% (wthol) testosterone in nutrient agar. Allantoin ic-screening program, strain A80407T (T = type strain) was decomposition was measured by the method of Kurup and isolated from soil collected in the Philippines. Strain Schmitt (5). NaCl tolerance was measured by streaking a A80407T produces a glycopeptide antibiotic of the ristocetin culture onto the surfaces of plates of ISP medium 2 contain- type which is not a lipoglycopeptide like ardacin, because it ing NaCl at different concentrations, and incubating the does not yield fatty acids upon hydrolysis. In this paper we preparations at 30°C for 14 days. The growth temperature describe strain A80407T and, based on its chemical proper- range was determined by inoculating culture plates of ISP ties and taxonomic characteristics, propose the name Kib- medium 2 and incubating the cultures at different tempera- delosporangium philippinense sp. nov. for this organism. tures for 7 to 14 days. Catalase, phosphatase, and urease were determined by methods of Blazevic and Ederer (2). MATERIALS AND METHODS Resistance to antibiotics. Antibiotic disks obtained from Difco Laboratories were placed onto the surfaces of ISP Bacterial strains. Strain A80407T was isolated from soil medium 2 agar plates that were seeded with a 2% inoculum collected in the Philippines by using selective isolation of the organism to be tested, and the preparations were procedures. Strain A80407.4 was derived from strain incubated at 30°C for 1 week. A80407T by N-methyl-N’-nitro-N-nitrosoguanidinemuta- Chemotaxonomy. Whole-cell hydrolysates prepared from tion. Kibdelosporangium aridum ATCC 39323T was pur- washed, lyophilized, 72-h vegetative growth were examined chased from the American Type Culture Collection, Rock- by using the methods of Becker et al. (1) and Lechevalier (8). ville, Md. Kibdelosporangium aridum subsp. largum ATCC Mycolic acid determinations were based on techniques de- 39922 was unavailable; therefore, previously published described by Minnikin et al. (13). Phospholipids were extracted scriptions (15) were used to obtain its characteristics. with CHGl,, chromatographed on Silica Gel 60 FZs4thin- layer chromatography plates in CHC1,-methanol-concen- * Corresponding author. trated NH20H (200:120: 15, vol, vol) against phospholipid

282 VOL. 38, 1988 KIBDELOSPORANGI UM PHILIPPINENSE SP. NOV. 283

TABLE 1. Taxonomic comparison of K. philippinense TABLE 2. Differentiating characteristics of K. philippinense and and K. aridum K. aridum subsp. largum ATCC 39922 Characteristic K. philippinense K. aridum K. aridum subsp. largum Characteristic philippinense K. ATCC 39922 Formatin of aerial mycelia Good Poor Formation of sporangiumlike Profuse Sparse NaCl tolerance (%) 7 structures Temp range (“C) 15-42 Reverse color Orange Off-white Peptonization of milk + yellow Hydrolysis of guanine + Soluble pigment production Occasional Rare Hydrolysis of allantoin + Crystal production - + Reduction of nitrate c Whole-cell wall components Utilization of Unknown spot with + - L- Arabinose + R(ribose)value of 0.15 Dextrin + Madurose + Glycogen + L-Rhamnose + a-Meth yl-D-glucoside + Utilization of Raffinose + L-Arabinose + Salicin + Dextrin + Glycogen + a-Meth yl-D-glucoside + D-Melezitose - standards, and visualized with a 10% ethanolic molybdo- D-Raffinose + phosphoric acid spray. Resistance to: Fatty acid analysis. Fatty acid methyl esters were analyzed Bacitracin (10 U) + by gas-liquid chromatography with a model 5898A comput- Gentamicin (10 p,g) + er-controlled gas-liquid chromatography system (Hewlett- - Oleandomycin (15 pg) Packard Co., Palo Alto, Calif.) (12). Lyophilized whole cells Streptomycin (10 pg) + grown for 72 h at 30°C were used for all fatty acid compar- NaCl tolerance (%) 27 Temp range (“C) 545 isons. Nitrate reduction - Protein analysis. Microbial fingerprints using [35S] Allantoin decomposition + methionine were generated and analyzed from polypeptide Guanine hydrolysis + banding patterns obtained by sodium dodecyl sulfate-poly- Peptonization of milk - + acrylamide gel electrophoresis. A computer-controlled sys- Glycopeptide antibiotic Ristocetinlike Ardacin tem developed by AMB Automated Microbiology Systems, produced Inc., San Diego, Calif., was used. Cultures were grown on Size of aerial spores (pm) 0.4 by 1.6 0.4 by Bennett agar (19) at 30°C for 72 h, and the proteins were 2.8 labeled with [35S]methionine,separated by electrophoresis, Size of sporangia (pm) 1-8 9-22 Fatty acid analysis and then visualized by beta scanning. The data generated 1~0-14:O(%) 13.4 2.8 were used to construct a dendrogram. Is0-16:O (%) 63.9 36.7 Scanning electron microscopy. A culture was fixed in Anteiso-15:O (%) 1.9 8.0 osmium tetroxide vapors for 20 h, dehydrated, rotary shad- trans-16: 1 (%) 4.1 owed with gold-palladium, and then viewed with an Etec Anteiso-17:1 (%) 11.0 scanning electron microscope.

RESULTS AND DISCUSSION Soil actinomycete strain A80407T showed the characteristics of the recently described genus Kibdelosporangium (14).

i~n~n7A m b A80407 I

W” Y Axis is % 16:lso FIG. 1. Fatty acid comparison of strains A80407T and A80407.4 and K. aridum. 284 MERTZ AND YAO INT. J. SYST.BACTERIOL.

Streptomyces coelicolor

Streptomyces lividans

Kibdelosporangium aridum

A80407 0.6, %,0.7 0.8 0.9 1.01480407.4

FIG. 2. Dendrogram showing protein polyacrylamide gel electrophoresis profile relationships among five actinomycete strains. Clus- tering of the coefficients of correlation was done by using the unweighted pair group method with average linkage.

Identity of genus. The new actinomycete genus Kibdelo- sporangium is defined by the presence of long chains of spores, as well as sporangiumlike structures, type IV cell walls (meso-diaminopimelic acid, D-glutamic acid, DL-ala- nine, muramic acid, N-acetyl-D-glucosamine, D-galactose, and arabinose) lacking mycolic acids, and a type A whole- cell sugar pattern (D-galactose and arabinose) plus traces of madurose (3-O-methyl-~-galactose).Strain A80407T has chemotaxonomic properties, cultural characteristics, and morphology that are consistent with its assignment to the genus Kibdelosporangium. No madurose was detected in whole-cell hydrolysates after several attempts; only trace levels of madurose are present in the genus according to the original Kibdelosporangium description (14). Identity of species. The genus Kibdelosporangium cur- rently contains one species, K. aridum, and one subspecies, K. aridum subsp. largum. Strain A80407T was compared with K. aridum type strain ATCC 39323 (B. A. Bowie, D. J. Newman, M. C. Shearer, R. D. Sitrin, and J. R. Valenta, U.S. patent 4,548,974, 22 October 1985). Cultural, morpho- FIG. 3. Scanning electron micrograph of spore chains of K. logical, chemical, and physiological differences are signifi- philippinense grown on ISP medium 4 for 3 weeks at 30°C. Bar = 1.0 cant. The antibiotic resistance patterns, carbon utilization wn. profiles, and fatty acid analyses of the two species are quite different (Table 1).K. aridum subsp. largum ATCC 39922 is media. Figure 1 is a three-dimensional plot showing the total unavailable at present for comparative studies. The previ- amounts of named fatty acids between 9 and 20 carbon ously published description of this subspecies (15) shows atoms long. Strains A80407T and A80407.4 were in a cluster significant differences with strain A80407T (Table 2). distinct from K. aridum. In addition, K. aridum has two fatty Fatty acid analysis was done on wild-type strain A80407T, acids that are not present in strains A80407T and A80407.4, mutant strain A80407.4, and K. aridum ATCC 39323T. Cells trans-16:l (4.1%) and anteiso-17:l (11%) acids (Table 1). of all three strains were grown for 72 h at 30°C in the same These differences, which generally should not occur within

TABLE 3. Cultural characteristics of K. philippinense

Color of reverse Aerial mycelium Medium at-owth Soluble pigment sides of colonieso Production Color ISP medium 2 Abundant 74. s.y Br Fair White Light brown ISP medium 3 Good 89.p.Y Good White None ISP medium 4 Abundant 89.p.Y Abundant White None ISP medium 5 Good 92.y White Fair White None ISP medium 7 Good 71.m.OY Fair White Very light brown Calcium malate Fair 70.1.0Y None None Czapek Good 89.p.Y Good White None Glucose-yeast extract agar Good 71.m.OY None Light reddish brown Glucose-as paraghe Good 71.m.OY Poor White None Nutrient agar Fair 71.m.OY Poor White None Tomato paste-oatmeal Abundant 77.m.yBr Good White to gray None agar Tap water agar Fair 92.y White Fair White None

a Color designations from reference 18. VOL.38, 1988 KIBDELOSPORANGIUM PHILIPPINENSE SP. NOV. 285

Many sporangiumlike structures were observed beneath these long spore chains (Fig. 4). They appeared to be borne apically on aerial hyphae or sporangiophores. These sporangiumlike structures were round, ranged from 1 to 10 pm in size, and had rugose surfaces (Fig. 5). No release of spores was observed. These structures had the appearance of true sporangia; however, since no release of spores was observed, they were neither true sporangia nor spore vesi- cles. Chemotaxonomy. Hydrolyzed whole cells of strain A80407T contained meso-diaminopimelic acid, galactose, glucose, mannose, arabinose, and ribose. An unknown pink- ish spot having an R(ribose)value of 0.15 was observed. No madurose was detected. Phosphatidylethanolamine was detected in whole-cell extracts. No mycolic acids were found. Thus, K. philippinense has type IV cell walls (lo), a type A sugar pattern (lo), and a type PI1 phospholipid composition (9). Physiological characteristics. K. philippinense produced acid from D-arabinose, cellobiose, a-methyl-D-glucoside, fructose, galactose, glucose, glycerol, inositol, lactose, maltose, melezitose, mannitol, mannose, melibiose, L-rhamnose, ribose, trehalose, and xylose. Acid was not produced from adonitol, L-arabinose, cellulose, destrin, dulcitol, ethanol, erythritol, glycogen, inulin, raffinose, salicin, sorbitol, sor- bose, sucrose, or xylitol. This organism utilized acetate, butyrate, citrate, formate, lactate, malate, oxalate, propio- FIG. 4. Scanning electron micrograph of numerous sporangium- nate, pyruvate, and succinate, but not benzoate or tartrate. like structures growing beneath the aerial hyphae. A culture was grown on ISP medium 4 for 3 weeks at 30°C. Bar = 10.0 pm.

the same species (M. Sasser, personal communication), suggest that K. aridum and strain A80407T are indeed two separate species. Protein polyacrylamide gel electrophoresis gives a pattern of polypeptide bands (17). This microbial fingerprint allows for computerized matching of protein patterns. A dendrogram was constructed with coefficients of correlation for wild-type strain, A80407T, mutant strain A80407.4, the K. aridum type strain, and two Streptomyces species (Fig. 2). Streptomyces coelicolor and Streptomyces lividans were included to illustrate a correlation coefficient at which species could be distinguished (i.e., an r value of 0.90 indicates separate species). Strains A80407T and A80407.4 clustered at an r value of 0.96, and K. aridum clustered at an r value of 0.81. These are ancillary data which further support the contention that strain A80407T and K. aridum are separate species. Cultural characteristics. K. philippinense grew slowly on complex and defined media. Aerial hyphae and sporangiumlike structures were produced on most media. Inorganic salts-starch agar (ISP medium 4) and tomato paste-oatmeal agar supported excellent substrate and aerial hypha development. The color of the aerial mycelium was white; the reverse color was generally pale yellow to orange yellow. SoIuble pigments were occasionally produced. The cultural characteristics of K. philippinense are summarized in Table 3. Neither crystal formation nor hyphal fragmentation as described previously for K. aridum (14) were observed with K. philippinense. Morphological characteristics. K. philippinense formed an extensive substrate mycelium and aerial hyphae with long FIG. 5. Scanning electron micrograph of one sporangiurnlike chains of spores. These spores were long cylindrical rods structure. A culture was grown on ISP medium 4 agar for 3 weeks at having smooth surfaces and measured 1.6 by 0.4 pm (Fig. 3). 30°C. Bar = 1.0 pm. 286 MERTZ AND YAO INT. J. SYST.BACTERIOL.

K. philippinense decomposed casein, calcium malate, 4. Gordon, R. E., D. A. Barnett, J. E. Handerhan, and C. H. Pang. elastin, esculin, hippurate, hypoxanthine, testosterone, ty- 1974. Nocardia coeliaca, Nocardia autotrophica, and the no- rosine, and urea. It did not decompose adenine, allantoin, cardin strain. Int. J. Syst. Bacteriol. 24:54-63. guanine, starch, or xanthine. 5. Kurup, P. Y., and J. A. Schmitt. 1973. Numerical taxonomy of K. philippinense produced catalase, H,S, and phospha- Nocardia. Can. J. Microbiol. 19:1035-1048. tase; it hydrolyzed milk, liquefied gelatin, and produced 6. Labeda, D. P. 1986. Transfer of “Nocardia aerocofonigenes” melanoid pigments on ISP medium 1 and ISP medium 6 but (Shinobu and Kawato 1960) Pridham 1970 into the genus Sac- charothrix Labeda, Testa, Lechevalier, and Lechevalier 1984 as not on ISP medium 7, tolerated levels of NaCl up to 2%, Saccharothrix aerocolonigenes sp. nov. Int. J. Syst. Bacteriol. grew in a temperature range from 20 to 40°C, was not 36:109-110. resistant to lysozyme, and reduced nitrate to nitrites. 7. Labeda, D. P., R. T. Testa, M. P. Lechevalier, and H. A. K. philippinense was resistant to 30 pg of cephalothin, 2 Lechevalier. 1984. Saccharothrix: a new genus of the Actinomy- pg of lincomycin, 15 pg of oleandomycin, 10 U of penicillin cetales related to Nocardiopsis. Int. J. Syst. Bacteriol. 34:42f% G, 10 pg of tobramycin, 30 pg of vancomycin, 30 p,g of 431. nalidixic acid, 300 U of polymixin B, 5 kg of trimethoprim, 8. Lechevalier, M. P. 1968. Identification of aerobic actinomycetes and 300 U of sulfadiazine. It was susceptible to 10 U of of clinical importance. J. Lab. Clin. Med. 71:934944. bacitracin, 10 pg of gentamicin, 30 pg of neomycin, 5 pg of 9. Lechevalier, M. P., C. DeBievi.e, and H. A. Lechevalier. 1977. rifampin, 10 kg of tetracycline, 30 kg of chloramphenicol, 15 Chemotaxonomy of aerobic actinomycetes: phospholipid com- pg of erythromycin, 30 pg of novobiocin, and 3 kg of position. Biochem. Syst. Ecol. 5:249-260. mandelamine . 10. Lechevalier, M. P., and H. Lechevalier. 1970. Chemical compo- Our comparisons indicated that strain A80407T is unlike sition as a criterion in the classification of aerobic actinomy- the previously described species of Kibdelosporangium. cetes. Int. J. Syst. Bacteriol. 20:435443. 11. Lechevalier, M. P., H. Prauser, D. P. Labeda, and J.-S. Ruan. Therefore, this strain is proposed as a new species, for which 1986. Two new genera of nocardioform actinomycetes: Arnyco- the name K. philippinense has been selected. lata gen. nov. and Amycofatopsis gen. nov. Int. J. Syst. Kibdelosporangium philippinense sp. nov. Kibdelospo- Bacteriol. 36:29-37. rangium philippinense (Phil. ip. pi. nen’. se. M.L. adj. 12. Miller, L., and T. Berger. 1985. Bacterial identification by gas philippinense, pertaining to the Philippines) cells are aero- chromatography and whole cell fatty acids. Gas chromatogra- bic, gram positive, non-acid fast, nonmotile, filamentous, phy application note 228-41. Hewlett-Packard Co., Palo Alto, and differentiated into substrate and aerial mycelia. Calif. Type strain. The type strain is strain A80407 (= NRRL 13. Minnikin, D. E., I. G. Hutchinson, A. B. Caldicott, and M. 18198). It was isolated from soil collected in the Philippines. Goodfellow. 1980. Thin-layer chromatography of methanoly- The species description is based on a single strain and thus sates of mycolic acid-containing bacteria. J. Chromatogr. serves as the type strain description. 188:221-233. 14I Shearer, M. C., P. M. Colman, R. M. Ferrin, L. J. Nisbet, and C. H. Nash 111. 1986. New genus of the Actinomycetales: ACKNOWLEDGMENTS Kibdelosporangium aridum gen. nov., sp. nov. Int. J. Syst. We thank Lee F. Ellis and Richard A. Schlegel, Eli Lilly & Co., Bac teriol. 36:47-54. Indianapolis, Ind., for their skillful technical assistance in obtaining 15. Shearer, M. C., A. J. Giovenella, S. F. Grappel, R. D. Hedde, the scanning electron micrographs. We thank Myron Sasser, Uni- R. J. Mehta, Y. K. Oh, C. H. Pan, D. H. Pitkin, andL. J. Nisbet. versity of Delaware, Newark, for the fatty acid analyses and Lynn 1986. Kibdelins, novel glycopeptide antibiotics. I. Discovery, Nye, AMB Automated Microbiology Systems, Inc., for the poly- production, and biological evaluation. J. Antibiot. 39:1386- peptide banding analyses. 1394. 16. Shirling, E. B., and D. Gottlieb. 1966. Methods for characteri- LITERATURE CITED zation of Streptomyces species. Int. J. Syst. Bacteriol. 16:313- Becker, B., M. P. Lechevalier, R. E. Gordon, and H. A. 340. Lechevalier. 1964. Rapid differentiation between Nocardia and 17. Tabaqchali, S., S. O’Ferrell, D. Holland, and R. Silman. 1984. Streptomyces by paper chromatography of whole-cell hydroly- Typing scheme for CIostridium dificile: its application in clini- sates. Appl. Microbiol. 12:421423. cal and epidemiological studies. Lancet i:935-938. Blazevic, D. J., and G. M. Ederer. 1975. Principles of biochem- 18. U. S. Department of Commerce National Bureau of Standards. ical tests in diagnostic microbiology. John Wiley & Sons, Inc., 1958. ISCC-NBS centroid color charts. Standard sample no. New York. 2106. U. S. Department of Commerce, Washington, D. C. Container Corporation of America. 1958. Color harmony man- 19. Waksman, S. A. 1961. The actinomycetes, vol. 2. The Williams ual, 4th ed. Container Corporation of America, Chicago. & Wilkins Co., Baltimore.