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Kibdelosporangium Philippinense Sp. Nov. Isolated from Soil

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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 de- scribed 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 characteris- tics of the recently described genus Kibdelosporangium (14). i~n~n7A m b A80407 I N 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 electro- phoresis 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)
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