Actinomycetologica (2009) 23:8–15 Copyright Ó 2009 The Society for Actinomycetes Japan VOL. 23, NO. 1 Isolation of Novel Actinomycetes from Spider Materials Kimika IwaiÃ, Susumu Iwamoto, Kazuo Aisaka and Makoto Suzukiy Innovative Drug Research Laboratories, Kyowa Hakko Kirin Co., Ltd., 3-6-6 Asahi-machi, Machida-shi, Tokyo, 194-8533, Japan (Received Oct. 20, 2008 / Accepted Mar. 12, 2009 / Published May 29, 2009) To collect new kinds of microorganisms for screening of biologically active substances, we focused on spider materials (webs, cuticle, egg sac), previously uninvestigated sources of such organisms. Using a new method of pre-treatment with 70% ethanol, 1,159 strains of actinomycetes were isolated from 196 spider materials, based on their morphological features. Of these, 293 strains were identified as non-filamentous actino- mycetes from their 16S rRNA gene sequences. More detailed examination indicated that 139 strains belonged to the suborders Micrococcineae, Frankineae and Propionibacterineae, and they included some novel strains of non-filamentous actinomycetes. Thus, spider materials provide a more useful source of non- filamentous actinomycetes than do soil samples. INTRODUCTION paper, we report a new method of isolation of micro- organisms from spider materials pre-treated with 70% The unique structural diversity inherent in natural ethanol, and we describe relationships between the kinds of products continues to be recognized for its value in the spider materials used and the taxonomic diversity of the drug discovery process (Fenical & Jensen, 2006). However, isolates obtained. there has been a recent decline in the rate of discovery of novel bioactive substances obtained from common terres- MATERIALS AND METHODS trial microorganisms, despite an increase in the rate of re- isolation of known compounds (Magarvey et al., 2004). To Types of spider materials circumvent this obstacle, new methods of isolation of A total of 196 kinds of spider materials were collected microorganisms have been extensively investigated. These from fields in the Kanagawa, Tokyo, Yamanashi and include the development of a humic acid-vitamin (HV) agar Okinawa prefectures in Japan. Spider materials were and other methods for the isolation of rare actinomycetes classified into ten types (Table 1). Typical ‘vertical spiral (Hayakawa et al., 1988, Hayakawa, 1990, 1994, 2003, orb webs’ (VO) and ‘horizontal spiral orb webs’ (HO) Hayakawa & Nonomura, 1993). Takahashi et al. (2003) were woven by spiders in several families (e.g. Araneidae, addressed the problem of oxidative stress in bacteria by the Tetragnathidae and Nephilidae). Orb webs are generally addition of superoxide dismutase and catalase to the isola- composed principally of sticky, capture-spiral silk, which tion medium, and succeeded in obtaining more colonies. is used to construct the central planar spiral. ‘Tangle-webs’ The discovery of useful actinomycetes from marine (TA) which are irregular in construction, and generally low sources has also been highlighted recently (Fiedler et al., and anchored to the ground or a wall, were woven by 2005) and at least three distinctive marine genera (Salini- spiders of the family Theridiidae. Flat platforms extending bacterium, Serinicoccus and Salinispora) have been de- from a funnel-shaped nest or ‘funnel-web’ (FU) were scribed. Fenical & Jensen (2006) reported the isolation of a woven by the Agelenidae and sheet weaver webs (SW) potent anticancer agent, salinisporamide A from Salinis- were constructed by the Linyphiidae. ‘Non-sticky threads’ pora tropica. Here we focus on a new natural environment, (NT) were obtained from Ariamnes cylindrogaster (Ther- distinct from the soil or the sea, as a potential source of idiidae). Although other species of web-building spiders do microorganisms that might yield novel secondary metab- not use their webs for prey capture directly, all spiders olites. We chose spider materials as isolation sources produce silk in the form of a ‘drag line’ (DL). Isolates from because they have not previously been exploited for this drag lines, obtained from spiders walking across the agar purpose and they are easy to collect. Furthermore, as plate, were included with samples taken from the surface of spiders typically prey on insects and other spiders, we the cuticle. Other categories of spider materials were hypothesized that they would be good sources of micro- obtained from the nest (NE), the molted cuticle or exuvia organisms that are associated with these prey items. In this (EC), and the egg sac (ES). ÃTo whom correspondence should be addressed. E-mail: [email protected], Phone: +81-042-725-2555, fax: +81-726-8330 yPresent address: Kyowa Hakko Bio Co., Ltd., 1-6-1 Ohtemachi, Chiyoda-ku, Tokyo 100-8185, Japan. Abbreviation: SEM; scanning electron microscopy. 8 ACTINOMYCETOLOGICA VOL. 23, NO. 1 Table 1. Spider materials from which isolates were obtained and (BacLightÔ, Molecular Probes, Inc.) and observed by their abbreviations used in the text. fluorescence microscopy (Axiophot 2, BP485/20 filter, Abbreviation Types of spider materials Carl Zeiss, Inc.). VO vertical orb web HO horizontal orb web Isolation and cultivation of microorganisms from spider materials TA tangle-web FU funnel-web Spider webs, captured with a spreader, were placed on et al. SW sheet weaver web HV agar (Hayakawa , 1987, 2000) which included NT non-sticky thread antibiotics (Cycloheximide 100 mg/L, Nystatin 50 mg/L, DL drag line Nalidixic acid 10 mg/L, Trimethoprim 20 mg/L). Alter- NE nest natively, webs were collected by inducing spiders to walk EC molted cuticle across the agar plate. The 9 cm diameter plate surface was ES egg sac then sprayed twice with 2 mL 70% ethanol to inhibit growth of other bacteria and fungi. The web was then dissolved in the ethanol and spread until dry. Plates were Microscopic observation of spider threads incubated aerobically at 28C for 3 weeks. All colonies that For scanning electron microscopy (SEM), spider threads appeared to be actinomycetes were picked out. Culture were spread onto carbon-tape, and coated with platinum- purification and cell biomass preparation were carried out palladium using an ion sputter coater (E-102, Hitachi, Ltd.), on KM398 medium (glucose 5 g, soluble starch 5 g, beef and observed in the SEM (S-570LB, Hitachi, Ltd.). Spider extract 1.5 g, yeast extract 2.5 g, tryptone peptone (DifcoÔ, threads were also stained using a bacterial viability kit BD) 2.5 g, MgSO4Á7H2O 0.5 g, KH2PO4 1g, Mg3(PO4)2Á Fig. 1. Scanning electron micrographs of the vertical orb web of Cyclosa kumadai. A; A thread which has been woven back and forth many times to form a bundle of increased strength. Scale bar, 176 mm. B; A rod-like cell measuring 1:35 mm  3:5 mm. This unfixed hydrated cell was deformed in the SEM. Scale bar, 2.5 mm. C; A small rod-like cell measuring 0:44 mm  0:9 mm. Scale bar, 1.5 mm.D; Multiple long rod cells (approx. 11 mm  3:7 mm) appear to be entangled in a thread. Scale bar, 17.6 mm. 9 ACTINOMYCETOLOGICA VOL. 23, NO. 1 Fig. 2. Observations of a fluorescence-dyed vertical orb web of Cyclosa kumadai. A; Drag line, Magnification, Â200. B; Capture-spiral silk, Magnification, Â200. C,D; Stabilimentum (web decorations), Magnification, Â2000. 8H2O 0.5 g, distilled water 1,000 mL, pH 7.2) at 28 C for 7 Dissolution of the lipid cell membranes of microorganisms to 14 days. Then the isolates were examined with a light is believed to take place in 70 w/w % (about 80 v/v %) microscope and categorized as either ‘non-filamentous’ or ethanol (Ingram, 1990). Gram-positive bacteria possess a ‘filamentous’ actinomycetes according to the absence or thick cell wall composed primarily of peptidoglycan (50– presence of a filamentous mycelium. The Gram reaction of 90%), whereas in Gram-negative bacteria this component is non-filamentous actinomycetes was determined using the smaller (10% of the cell wall) and an additional lipid- method of Buck (1982). Isolates were preserved in 20% containing outer membrane is present (Beveridge, 1990, glycerol solution at À80C. PCR amplification of 16S Salton et al., 1996). Thus, Gram-negative bacteria would be rRNA genes of isolates was carried out as described by expected to be more easily disrupted by 70% ethanol than Tanner et al. (2000). The amplified fragments were purified Gram-positive bacteria. Although fungal colonies some- by MagExtractor (Toyobo Co, Ltd.). Sequencing was times grew on the agar, even after treatment with 70% carried out with a BigDye Terminator Cycle Sequencing ethanol, provided inhibition was almost complete, actino- FS Ready Reaction kit and an ABI 377 DNA sequencer mycete colonies were able to develop adequately and their (Applied Biosystems, Inc.). colonies were easily isolated. This procedure thus provides a relatively simple and effective method for the isolation of RESULTS AND DISCUSSION actinomycetes from spider material. Microscopic observation of the spider threads Correlation of spider materials and taxonomic groups In the SEM, we observed cell-like materials adhering to of isolates spider threads (Fig. 1). Although the mycelia could not be Samples of spider materials used for isolation of actino- identified, the fluorescent dye confirmed that these were mycetes were divided into ten categories according to live microorganisms (Fig. 2). Cells were observed on the morphological characteristics. Actinomycetes isolated from surface of the threads but they were not present internally. spider materials were classified down to suborder level and Orange/red-stained dead cells were also observed. Black were further grouped as filamentous or non-filamentous. spots were assumed to be inorganic material. From their morphology observed under the microscope, 1,159 strains of actinomycetes were identified in isolates Pre-treatment of spider materials with 70% ethanol from 196 spider materials. Of these, 293 strains were Without the ethanol pre-treatment, the isolation medium identified by 16S rRNA gene sequencing as non-filamen- was covered with fungi and Gram-negative bacteria.