APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1999, p. 1930–1935 0099-2240/99/$04.00ϩ0 Copyright © 1999, American Society for Microbiology. All Rights Reserved. Vol. 65, No. Selective Isolation and Distribution of Sporichthya Strains in Soil SHIN-ICHI SUZUKI,* TORU OKUDA, AND SABURO KOMATSUBARA Screening Research Laboratory, Discovery Research Laboratories, Tanabe Seiyaku Co., Ltd., 2-2-50 Kawagishi, Toda-shi, Saitama 335-8505, Japan Received 16 November 1998/Accepted 17 February 1999 microscope lens with a long working distance is an effective way to examine plates (30). Takeuchi et al. have isolated Sporichthya strains from soil samples collected in Japan by centrifugation (27). To our knowledge, no other Sporichthya isolates have been described. Therefore, we focused on the genus Sporichthya as a new screening source of bioreactive metabolites, and in this paper we describe a selective isolation method. Hayakawa and Nonomura described a medium, humic acidvitamin agar (HVA), that contains soil humic acid as the sole source of carbon and nitrogen (7). This medium yields a large number of actinomycete colonies on isolation plates and supports good sporulation; hence, we used HVA as a basal medium. Gellan gum, a polysaccharide produced by Pseudomonus elodea, is used for plant tissue culture as a solidifying agent because it stimulates growth. In a previous study, we examined the effect of this solidifying agent on spore formation in Actinobispora yunnanensis IFO 15681 and found that gellan gum plus calcium chloride significantly stimulated the formation of spores and aerial mycelium (26). In this study, we used gellan gum plus calcium chloride in our Sporichthya isolation medium. On the basis of the Sporichthya strains isolated by the method described below, we discuss ecological aspects of the genus. The actinomycetes are one of the most attractive sources of new bioactive metabolites. However, the rate of discovery of new compounds has decreased, since the ubiquitous species have been studied extensively. New species have the potential to produce new metabolites, which justifies the isolation of new species at pharmaceutical research laboratories (25). Selective isolation methods have, therefore, been developed for taxa that are seldomly isolated by conventional dilution plate methods (8–14, 17, 28). The genus Sporichthya is characterized by a gram-positive type of cell wall, a facultatively anaerobic nature, and a cell wall containing a large amount of L-diaminopimelic acid. Sporichthya aerial hyphae are initiated upright on the surface of the medium by holdfasts, which are outgrowths of the wall of the basal cell. Primary mycelium is not formed. The sparingly branched aerial mycelium contains rod-shaped to coccoid spores, which may become polarly flagellated and motile in the presence of water (30). The genus Actinoplanes, another zoosporic actinomycete genus, was first described by Couch in 1950 (2). Selective methods, such as pollen or keratin baiting techniques (3, 5, 11, 16, 22), chemotactic methods (5, 10, 13, 23–24), and centrifugation methods (18, 21, 27), have been developed to isolate zoosporic actinomycetes that are difficult to isolate by conventional dilution plate methods. The monospecific genus Sporichthya was first described by Lechevalier et al. in 1968 (20). Sporichthya polymorpha was isolated from a sample of greenhouse soil collected at Rutgers University in New Jersey. The genus Sporichthya appears to be a rare taxon, since only five strains have been isolated in the laboratory of Lechevalier et al. during the past 20 years. Sporichthya colonies growing on tap water agar or Czapek agar diluted 1/10 are hard to recognize by eye. A ϫ10 to ϫ40 MATERIALS AND METHODS Microbial strains. S. polymorpha KCC A0089T (ϭ IFO 12702T) was obtained from Kaken Chemical, Tokyo, Japan. Sporichthya sp. strains AS 1734, AS 1972, and AS 2345 were isolated in our laboratory from soil samples BS793, BS665, and BS1126, respectively. Soil samples. A total of 466 soil samples were collected in 28 countries from 1993 to 1996. The samples were passed through a 0.71-mm mesh sieve after they were air dried at room temperature for to 14 days and were stored at room temperature in plastic bags until they were used. Soil samples BS793, BS665, and BS1126 were collected in the United Kingdom, the United States, and Taiwan, respectively. Soil pH measurement. One gram of a soil sample was suspended in ml of distilled water and mixed well. After 30 s of standing, the supernatant was used to determine the pH. Another ml of water was added if the supernatant was not recovered. * Corresponding author. Mailing address: Screening Research Laboratory, Discovery Research Laboratories, Tanabe Seiyaku Co., Ltd., 2-2-50 Kawagishi, Toda-shi, Saitama 335-8505, Japan. Phone: 81-48433-2564. Fax: 81-48-433-2565. E-mail: s-suzuki@tanabe.co.jp. 1930 Downloaded from http://aem.asm.org/ on June 30, 2015 by guest A simplified enrichment method in which centrifugation is used for selective isolation of Sporichthya strains from soil is described. Gellan gum plus mM CaCl2 stimulated growth of Sporichthya polymorpha KCC A0089 so that this organism was readily recognized on an isolation plate. High yields of motile spores were obtained by using a flooding solution containing 0.1% skim milk in 10 mM MOPS (morpholinepropanesulfonic acid) (pH 8.0) and then incubating the preparation at 27°C for 60 and centrifuging it at 1,000 ؋ g for 10 min. Dry heat treatment at 80°C for 60 increased the ratio of Sporichthya colonies to nonfilamentous bacteria on a gellan gum plate. Since S. polymorpha was sensitive to 14 antibiotics, including nalidixic acid, addition of these antibiotics was not suitable for isolating Sporichthya strains. Our isolates were identified as Sporichthya strains on the basis of their morphological and chemotaxonomic characteristics. By combining the techniques described above, we isolated a number of Sporichthya strains from 21 soil samples, which were collected in Belgium, France, India, Japan, Papua New Guinea, Spain, Taiwan, the United Kingdom, and the United States. Sporichthya strains are widely distributed in the world. To our knowledge, this is the first time that Sporichthya strains have been isolated from locations other than the United States or Japan. VOL. 65, 1999 ISOLATION AND DISTRIBUTION OF SPORICHTHYA STRAINS 1931 Media. Humic acid-vitamin-gellan gum medium (HVG), which was based on HVA (7), contained 0.05% humic acid, mM CaCl2, and 0.7% gellan gum (Wako Pure Chemicals, Osaka, Japan). The following media, with slight modifications, were also used in the comparative study: colloidal chitin agar (15), starch-nitrate-casein agar (19), actinomycete isolation agar (4), and egg albumin agar (29). The pH of each medium was adjusted to 7.2, and the media were autoclaved at 121°C for 20 and cooled to 55°C. A membrane filter-sterilized solution of cycloheximide in diluted ethanol was added aseptically to the medium to give a final cycloheximide concentration of 50 g/ml. HVG plates were cooled to room temperature on a clean bench immediately after the medium was poured. Effect of flooding on Sporichthya isolation. Soil samples from which Sporichthya strains were isolated were used to examine the effect of flooding on Sporichthya isolation; no dry heat treatment was included. Effect of flooding on Sporichthya motility. Four Sporichthya strains, including the type strain, were grown on HVG plates at 27°C for 14 days to obtain maximum sporulation. Spores of the Sporichthya strains were added to 200 l of a preincubated flooding solution in a 96-well microtiter plate. After incubation, motility was checked with a phase-contrast microscope (magnification, ϫ200) by using a hemocytometer (Thoma). Antibiotic susceptibility of S. polymorpha. Antibiotics (adriamycin, amikacin, bacitracin, daunorubicin, erythromycin, kanamycin, leucomycin, minocycline, nalidixic acid, novobiocin, paromomycin, rifampin, tunicamycin, and vancomycin) were diluted with 1% tryptone to obtain final concentrations of 0.78 to 100 g/ml by using 96-well microtiter plates. A loopful of well-sporulated S. polymorpha cells was mixed with ml of sterile saline, and 20 l of the spore suspension was added to 80 l of an antibiotic solution in a microtiter plate. The microtiter plates were incubated at 27°C for 48 h. MIC were determined visually on the basis of Sporichthya growth. Selective isolation of Sporichthya strains from soil. Air-dried soil samples (200 mg) were gently ground and kept in a hot-air oven at 80°C for 60 min. After the soil samples were cooled at room temperature, they were added to 2-ml portions of a flooding solution containing 0.1% skim milk (Difco) in 10 mM MOPS (morpholinepropanesulfonic acid) (pH 8.0). Each soil suspension was incubated at 27°C for 60 with occasional stirring to stimulate motility. After the soil suspension was centrifuged (1,000 ϫ g, 10 min, room temperature), 100 to 500 l of supernatant was gently transferred to a sterile tube. A zoospore suspension that was serially diluted with sterile saline was spread onto HVG plates. The plates were incubated at 27°C for 21 to 28 days. Actinomycete colonies were observed directly with a light microscope equipped with a ϫ40 long-workingdistance objective lens. Sporichthya colonies identified on the basis of their morphological characteristics were purified by single-colony isolation. Electron microscopic observation. Cultures grown on HVG at 27°C for 21 to 35 days were observed with a Hitachi model S-4200 scanning electron microscope (SEM). Samples were exposed to 2% OsO4 vapor in situ at room temperature for h, mounted on specimen stubs with carbon tape, and coated with gold. Chemotaxonomic characterization. For chemotaxonomic analysis, the strains were cultured at 27°C for 21 days on HVG plates. Whole-cell hydrolysates were prepared by the method of Becker et al. (1). Diaminopimelic acid isomers were analyzed by thin-layer chromatography as described by Hasegawa et al. (6). TABLE 1. Effects of solidifying agents and bivalent cations on Sporichthya growth Bivalent cation CaCl2 MgCl2 ⅐ 6H2O MgSO4 ⅐ 7H2O CaCl2 a Solidifying agent Gellan gum Gellan gum Gellan gum Agar Growth at a cation concn of: mM mM a ϩ ϩϩϩ ϩϩ ϩϩ ϩ mM 10 mM ϩϩ ϩϩ ϩ ϩ ϩ ϩ ϩ ϩ ϩϩϩ, good growth; ϩϩ, moderate growth; ϩ, poor growth. Downloaded from http://aem.asm.org/ on June 30, 2015 by guest FIG. 1. SEM micrographs (a through c) and light micrograph (d) of Sporichthya strains grown on HVG for 35 days at 27°C. (a) S. polymorpha KCC A0089T. Bar ϭ m. (b) Sporichthya sp. strain AS 1972. Bar ϭ m. (c) Sporichthya sp. strain AS 2345. Bar ϭ m. (d) Sporichthya sp. strain AS 1734. Bar ϭ 50 m. 1932 SUZUKI ET AL. APPL. ENVIRON. MICROBIOL. TABLE 2. Effects of flooding solutions on Sporichthya isolation No. of Sporichthya colonies (102 CFU/g of soil) Time (min) a Flooding solution Sample BS665 Sample BS793 Sample BS1126 Avg 30 60 16 24 61 0 11 15 43 73 10 22 48 1% Beef extract 30 60 40 12 15 32 39 91 24 17 36 1% Tryptone 30 60 16 13 0 25 21 10 12 0.1ϫ Potato-carrot broth 30 60 27 11 16 15 10 mM MOPS (pH 7.0) 30 60 13 0 4 Distilled water 30 60 19 1 17 16 12 a Compounds were dissolved in 10 mM MOPS (pH 7.0). grew slowly on HVG plates and developed white colonies after incubation for 28 days at 27°C. Although the colors of the colonies were almost identical, the growth rates on various media differed from strain to strain. SEM observation revealed that abundant spore chains were produced (Fig. 1). These spore chains were borne directly on the basal cells on the medium and branched several times. The spores were spherical or cylindrical and had smooth surfaces. Substrate mycelia were not observed with a light microscope. TABLE 3. Effects of temperature of the flooding solutiona Time (h) Strain T FIG. 2. Growth of S. polymorpha KCC A0089T on HVG containing mM CaCl2 (a), HVG containing mM MgCl2 ⅐ 6H2O (b), and HVA (c). The plates were incubated for 14 days at 27°C. RESULTS AND DISCUSSION Morphological and chemotaxonomic characterization of Sporichthya isolates. Three typical isolates, AS 1734, AS 1972, and AS 2345, were characterized taxonomically. These strains % of motile spores at: 10°C b 20°C 27°C 32°C 45°C Ϫ Ϫ ϩ ϩϩ ϩ ϩ ϩ ϩϩ ϩ ϩ ϩ ϩϩ Ϫ Ϫ Ϫ Ϫ S. polymorpha KCC A0089 Ϫ Ϫ Ϫ Ϫ Sporichthya sp. strain AS 1734 ϩ Ϫ ϩ ϩ ϩϩ ϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩ ϩ ϩ ϩ Ϫ Ϫ Ϫ Ϫ Sporichthya sp. strain AS 1972 ϩ Ϫ ϩ ϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩϩ Ϫ Ϫ Ϫ Ϫ Sporichthya sp. strain AS 2345 ϩ Ϫ ϩ ϩ ϩϩ ϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩ Ϫ Ϫ Ϫ a b The flooding solution used was 0.1% skim milk in 10 mM MOPS (pH 7.0). Ϫ, Ͻ10%; ϩ, 10 to 50%; ϩϩ, Ͼ50%. Downloaded from http://aem.asm.org/ on June 30, 2015 by guest 0.1% Skim milk ISOLATION AND DISTRIBUTION OF SPORICHTHYA STRAINS VOL. 65, 1999 1933 TABLE 4. Effects of pH of the flooding solution on Sporichthya isolationa Soil sample BS665 BS793 BS1126 a No. of Sporichthya colonies (102 CFU/g of soil) at: pH pH pH pH pH 19 68 37 72 33 131 63 215 49 51 The flooding solution used was 0.1% skim milk in 10 mM MOPS. TABLE 5. Effect of dry heating on isolation of Sporichthya spp. No. of colonies (102 CFU/g of soil) a b 35 (32.7)b 63 (69.9) 57 (75.9) 36 (66.7) Nonfilamentous bacteria Actinomycetesa Sample BS1126 Sporichthya spp. Nonfilamentous bacteria Actinomycetesa Sample BS793 Sporichthya spp. Nonfilamentous bacteria Actinomycetesa Length of dry heating at 80°C (h) Sporichthya spp. Sample BS665 19 54 11 (23.9) 29 39 (43.1) 12 40 17 10 15 (33.8) 11 17 129 (87.7) 11 12 11 (39.0) 11 57 (73.5) 13 13 (34.5) 47 (86.4) Actinomycetes other than Sporichthya spp. The values in parentheses are percentages. FIG. 3. Sporichthya colonies on a selective isolation plate. The soil sample used was sample BS665. The arrows indicate Sporichthya colonies. Sporichthya strains by using the soil samples from which Sporichthya strains were isolated (Table 2). Four times more Sporichthya colonies were obtained when we used 0.1% skim milk containing 10 mM MOPS (pH 7.0) than when we used distilled water. The skim milk concentration had no effect on motility, and optimal motility was observed at 27°C (Table 3). Most strains exhibited good motility at neutral to slightly alkaline pH values, and the optimum pH was 8.0 (Table 4). Tap water, 10% soil extract in 10 mM phosphate buffer (13), yeast extract solutions, and sucrose solutions are commonly used as flooding solutions. More motile spores were obtained with 0.1% skim milk (nonsterilized) than with other media when these preparations were used as flooding solutions for S. polymorpha. However, the ratio of motile spores to nonmotile spores decreased when the skim milk solution was autoclaved, indicating that substances that stimulate Sporichthya motility are sensitive to high temperature and/or pressure. Motile spores were obtained immediately after Sporichthya spores were exposed to the flooding solution. This behavior is different from the behavior of zoosporic actinomycetes that produce sporangiospores; in Actinoplanes italicus, Planobispora longispora, and Planomonospora parontospora cultures it takes 20 to 60 for motility to occur. A flooding solution containing 0.1% skim milk also efficiently stimulated motility of zoosporic actinomycetes other than Sporichthya strains (data not shown). In other words, skim milk was an effective agent for selectively isolating zoosporic actinomycetes by the centrifugation method. Antibiotic susceptibility of S. polymorpha. Specific antibiotics are commonly used to selectively isolate members of a particular actinomycete genus. S. polymorpha was weakly resistant to nalidixic acid (MIC, 25 g/ml), yet was susceptible to 13 other antibiotics. However, other Sporichthya isolates were sensitive to to 20 g of nalidixic acid per ml. The sensitivity to antibiotics is probably one of the reasons why Sporichthya strains are rarely isolated, because various antibiotics are often added to the isolation media used for rare actinomycetes. Effect of dry heat treatment. During selective isolation of actinomycetes, reducing the number of nonfilamentous bacteria on the isolation medium is important. Heat treatment of soil is another method for inhibiting bacterial growth. As Downloaded from http://aem.asm.org/ on June 30, 2015 by guest The diaminopimelic acid analysis revealed that the isomer present was the L isomer, as was the case for the type strain of S. polymorpha, which indicated that the cell wall type was type I. Our isolates were, therefore, identified as Sporichthya strains on the basis of their morphological and chemotaxonomic characteristics. Effect of isolation media on Sporichthya growth. Since S. polymorpha grew poorly on tap water agar or Czapek agar diluted 1/10, it was difficult to identify this organism by eye (30). Formation of large colonies is helpful for rapid recognition of isolates. Suzuki et al. (26) developed HVG containing calcium chloride and gellan gum as a solidifying agent for selective isolation of Actinobispora strains. HVG significantly stimulated the formation of aerial mycelia by Actinobispora yunnanensis IFO 15681. Various media were tested, including colloidal chitin agar, actinomycete isolation agar, egg albumin agar, and HVA, and HVG, and HVG produced optimal Sporichthya growth (data not shown). Because a bivalent cation (2 to 15 mM) is indispensable for solidifying gellan gum solutions, we examined the influence of various cations on the growth of S. polymorpha (Table 1). A combination of gellan gum and mM CaCl2 was essential for good growth of S. polymorpha. In contrast, when MgCl2 or MgSO4 was used with gellan gum or when CaCl2 was used with agar, much less growth occurred (Fig. 2). Effects of flooding solutions on motility. Development of a flooding solution which recovered a high percentage of the motile spores led to successful selective isolation of zoosporic actinomycetes by the centrifugation method, as described by Makker and Cross (21) and Takeuchi et al. (27). As a result of a preliminary examination, high percentages of motile spores were recovered with skim milk (nonsterilized), beef extract, tryptone, or 0.1ϫ potato-carrot broth (data not shown). We examined the effects of flooding solutions on recovery of 1934 SUZUKI ET AL. APPL. ENVIRON. MICROBIOL. TABLE 6. Distribution of Sporichthya strains No. of soil samples (pH range) Continent Country From which Sporichthya sp. was isolated Tested % of positive samples Egypt Tanzania Uganda 10 (7.7–9.2) (4.8–8.0) (6.5–7.4) 0 Asia India Indonesia Japan Malaysia Myanmar Singapore Taiwan 22 (6.9–8.5) 29 (4.4–7.7) 87 (3.9–7.9) 14 (3.6–6.5) (6.8–7.2) (4.7–6.9) 11 (4.7–8.4) (7.9) (6.3–7.3) 0 (7.1–8.0) 5 0 18 Europe Austria Belgium Denmark France Greece The Netherlands Spain United Kingdom (5.3) (3.5–7.3) 13 (4.7–8.6) 35 (5.3–8.3) (7.8) (5.2) (7.1) 15 (4.5–8.1) (7.0) (7.0–7.7) 0 (7.1) (7.3–7.7) 14 11 0 100 20 North America Canada United States 11 (5.3–8.2) 34 (4.5–8.3) (6.1–7.8) Oceania Australia Papua New Guinea Solomon Islands 70 (4.1–9.2) (6.0–7.2) (7.2–7.9) (6.8) 14 South America Argentina Bolivia Brazil Ecuador Paraguay 16 (5.2–8.5) 24 (4.2–8.1) 16 (4.9–7.6) (7.1–7.7) (5.5–7.3) (7.2) (6.4) 0 0 Total 466 (3.5–9.2) shown in Table 5, S. polymorpha was resistant to dry heating at 80°C for to h. Selective isolation of Sporichthya strains from soil. HVG plates, a flooding solution containing 0.1% skim milk in 10 mM MOPS (pH 8.0), and dry heating at 80°C for 60 were used to isolate Sporichthya strains from 466 soil samples collected in 28 countries (Table 6). The gellan gum medium was more transparent than agar media, so relatively large colonies were readily visible on the medium (Fig. 3). Sporichthya-like isolates were obtained from 21 of the 466 samples (4.5%); isolates were obtained from samples collected in Argentina, Belgium, Bolivia, France, India, Japan, Papua New Guinea, Spain, Taiwan, the United Kingdom, and the United States. These isolates resembled each other in terms of their morphological characteristics, which were similar to the characteristics described by Lechevalier et al. (20) for S. polymorpha, although the growth rates on various media differed from strain to strain. Sporichthya strains were isolated from soil samples with pH values ranging from 6.1 to 8.0, which suggests that Sporichthya strains prefer a neutral-pH environment. Our findings revealed that Sporichthya strains are widely distributed in Asia, Europe, North America, Oceania, and South America. To our knowledge, this is the first time that Sporichthya strains have been found in locations other than the United States or Japan. 21 (6.1–8.0) 0 4.5 ACKNOWLEDGMENTS We thank Kyoko Kato and Yuka Hiruma for technical assistance. REFERENCES 1. Becker, B., M. P. Lechevalier, R. E. Gordon, and H. A. Lechevalier. 1964. Rapid differentiation between Nocardia and Streptomyces by paper chromatography of whole-cell hydrolysates. Appl. Microbiol. 12:421–423. 2. Couch, J. N. 1950. Actinoplanes, a new genus of the Actinomycetales. J. Elisha Mitchell Sci. Soc. 66:87–92. 3. Couch, J. N. 1954. The genus Actinoplanes and its relatives. Trans. N. Y. Acad. Sci. 16:315–318. 4. Difco Laboratories. 1962. 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MICROBIOL. on June 30, 2015 by guesthttp://aem.asm.org/Downloaded from The diaminopimelic. desert soils. J. Ind. Microbiol. 17:260–267. 6. Hasegawa, T., M. Takizawa, and S. Tanida. 1983. A rapid analysis for chemical grouping of aerobic actinomycetes. J. Gen. Appl. Microbiol. 29: 319–322. 7.