The taxonomic list of termite genera from Pearce et al.6) Ter mitidae family is ... of other cellulolytic bacteria prevented the isolation ... each species lived longest.
Biosci. Biotechnol. Biochem., 67 (8), 17971801, 2003
Note
Isolation of Actinomycetes from Termites' Guts Yoshio WATANABE,1,õ Naoya SHINZATO,2 and Takema FUKATSU 2 1Bioresource
Laboratories, Mercian Corporation, 1808 Nakaizumi, Iwata, Shizuoka 4380078, Japan for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology, Tsukuba Central 64A, 111 Higashi, Tsukuba, Ibaraki 3058566, Japan
2Institute
Received June 17, 2002; Accepted April 29, 2003
Actinomycetes could be isolated eciently on a defat ted wood powder medium from the guts of various species of termites. The actinomycete ora in the termites' guts depended largely on the area in which the termites naturally occur. In termites from the same area, the actinomycete ora changed depending on the taxonomic dierence between termite species. Some actinomycetes isolated from termites' guts grew satis factorily on ligninrelated media, and the others grew on celluloserelated media. Key words:
actinomycetes; termite gut; wood powder medium; isolation method
In 1979, Bignell et al., using an electron micro scope, observed actinomycetelike bacteria in the guts of termites.1) Actinomycetes were isolated from termites' guts by Pasti and Belli in 1985, and were found to have cellulolytic activity.2) These actinomy cetes were later also found to have ligninsolubilizing activity.3,4) Avicel was used as a carbon source for the isolation, but in many cases the predominant growth of other cellulolytic bacteria prevented the isolation of pure actinomycetes. Termites can live solely on wood for food. When a medium containing only Japanese beech wood powder with the fat removed was used for the isolation of microorganisms, the dominant colonies were of lamentous actinomycetes rather than other bacteria. Ramachandra et al. reported characteriza tion of the lignin degradation enzyme from the actinomycete Streptomyces viridosporus isolated from soil as a microorganism which had lignin degra dation activity.5) In this paper, we report a novel medium and method for the isolation of actinomy cetes from the guts of termites. The taxonomic positions of the termites used in this study are shown in Fig. 1. Coptotermes formosa nus were collected from the islands of Yakushima and Iriomotejima in the southwest of Japan, and were also taken from termite colonies kept at the Forestry and Forest Products Research Institute (FFPRI) for more than 15 years (Kentaro Suzuki õ
Fig. 1. Taxonomic Position of Termites Used in This Study. The taxonomic list of termite genera from Pearce et al.6) Ter mitidae family is the only higher order of termites which do not have protozoa in the guts, the other families are the lowerorder termites, which have protozoa in the guts. As the lowerorder termites mainly inhabit Japan, all genera are shown on them, on the other hand subfamily and the numbers of genera consisting of the subfamily are shown on the higherorder termites.
Strain, Miyazaki Prefecture origin). Reticulitermes speratus were collected from the island of Yonagunijima and Tsukuba (from the ground of National Institute of Advanced Industrial Science
To whom correspondence should be addressed. Fax: {81538211135; Email: watanabeysomercian.co.jp
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Y. WATANABE et al. Table 1.
Isolation of Actinomycetes from Coptotermes formosanus Guts*
Medium
Number of colonies appearing (colonies W individual) Filamentous actinomycetes
Other bacteria
Defatted wood powder (Iriomotejima termites) Defatted wood powder (Yakushima termites) Dealkaline lignin HVA7)
307 361 67 61
8 40 167 230
ISP2 8,9) Avicel Cellulose powder Carboxymethyl cellulose (CMC) Nutrient broth
158 25 11 0 0
3505 930 1923 365 13500
Ratio (Filamentous actinomycetes W Other bacteria) 38 9.0 0.40 0.27 0.045 0.027 0.0057 0 0
* Iriomotejima termites were used except where otherwise mentioned.
and Technology, AIST). Neotermes koshunensis and Odontotermes formosanus were collected from Iriomotejima, and Hodotermopsis japonica were collected from Yakushima. Defatted wood powder was prepared by subjecting Japanese beech powder to Soxhlet extraction with ethanol:toluene (2:1) for 24 h, and then drying in a draft chamber. The defatted wood powder medium comprised defatted Japanese beech powder (2z) and agar (1.5z) in distilled water. For the isolation of microorganisms in the termites' guts, several other types of media were used in addition to the defatted wood powder medium. These were dealkaline lignin medium (0.1z dealkaline lignin, Tokyo Kasei Co. (Tokyo), pH 7.0), HVA medium (0.1z humic acid, Wako Pure Chemical Industries (Osaka), 0.05z Na2 HPO4¥12H2 O, 0.17z KCl, 0.001z FeSO4¥ 7H2 O, and 0.001z CaCO3, pH 7.0; after autoclaving (1219C, 15 min) a ltersterilized vitamin solution (0.005z thiamin hydrochloride, 0.005z riboavin, 0.005z nicotinic acid, 0.005z pyridoxine hydrochloride, 0.005z inositol, 0.005z pantothenic acid calcium salt, 0.005z paminobenzoic acid, and 0.0025z biotin) was added),7) ISP2 medium (Nihon Pharm. Co. (Tokyo), Actino Medium No. 2),8,9) and nutrient broth (Kyokuto Pharm. Co. (Tokyo)). Celluloserelated media used were Avicel medium (2z Avicel, pH 7.0), cellulose powder medium (2z cellulose powder, pH 7.0), and carboxymethyl cellulose (CMC) medium (2z CMC, pH 7.0). The agar concentration of each medium was 1.5z. The guts were pulled out from 20 mg of termites of each species and suspended in 1 ml of saline. The gut contents were pressed out with tweezers on a clean bench, mixed, and plated. The concentrations of saline used were 0.4z for C. formosanus, R. spera tus, and O. formosanus, and 0.6z for N. koshunen sis and H. japonica, in which protozoa in the gut of each species lived longest. The number of microbes per individual termite was evaluated after the colo nies that appeared on the plates had been transferred
to a slant (200300 random colonies) and growth on the slant had been checked for sameness with the naked eye. The `Guide to Color Standard' 10) was used to identify mycelial colors. DNA was extracted from actinomycete cells for identication as follows: The actinomycete cells were collected from the culture broth for 2 days at 309C in ISP2 liquid medium with centrifugation for 5 min at 5,000 rpm. The cells were suspended in 360 ml of TE buer (10 mM TrisHCl, 1 mM EDTA; pH 8) and repeatedly frozen and thawed 2 times. Then 40 ml of 10 mg W ml lysozyme (nal concentration 1 mg W ml) was added and incubated for 30 min at 509 C. Furthermore, 100 ml of 10 mg W ml proteinase K (nal concentration 1 mg W ml) and 500 ml of BL buer (40 mM TrisHCl, 1z Tween 20, 0.2z Nonidet P40, 0.2 mM EDTA; pH 8) were added and incubated for 30 min. Proteinase K was inactivated by incubation for 10 min at 909 C and then 1 ml of the reaction mixture was put through PCR. The actinomycete 16S rDNA was amplied by PCR. The PCR primers used were Eub11f3mx (TGRGTTTGATCMTGGCTYAG; E. coli positions 8 to 27) and Eub1511r1mx (TGGHTACCTTGTTA CGACTT; positions 1492 to 1511), which were designed to selectively amplify 16S rDNA of the domain Bacteria. PCR was done in 50 ml of reaction mixture (10 mM TrisHCl; pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 50 mM each deoxynucleoside tri phosphate, 10 pmol each primer, 2.5 U Taq DNA polymerase [TaKaRa Taq, Takara Shuzo Co., Ltd., Japan]) by using the following temperature cycling conditions: initial denaturation at 949C for 3 min, 20 cycles of 949 C for 30 s, 539C for 30 s, and 729C for 1.5 min, and a nal extension at 729 C for 10 min. The PCR products were puried using spin columns (Qiagen, see above), then used for the sequencing reaction. Sequences were analyzed bi directionally with the primers Eub906f1mx (AAAC TCAAAGGAATTGRCGG), and Eub1405r1mx (GACGGGCGGTGTGTRCA). The DNA sequence
Isolation of Actinomycetes from Termites' Guts Table 2.
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Microorganisms from the Guts of Various Termite Species Isolated on Defatted Wood Powder Medium Number of colonies appearing (colonies W individual)
Termite species Coptotermes formosanus (Average weight 3.8 mg W individual) from Yakushima from Iriomotejima kept in FFPRI * Reticulitermes speratus (Average weight 1.8 mg W individual) from Yonagunijima from Tsukuba (AIST ** ) Neotermes koshunensis (Average weight 23 mg W individual) from Iriomotejima Hodotermopsis japonica (Average weight 24 mg W individual) from Yakushima Odontotermes formosanus (Average weight 1.8 mg W individual) from Iriomotejima
Filamentous actinomycetes (Type ratio *** )
361 (CA1:CA2:CA3:CA4155:29:166:11) 307 (CA1:CA2123:184) 0
(CA1:CA2499:114)
Other bacteria
Fungi
40
0
8
0
717
141
613
93
0
0
13
0
1446
0
276
0
12
0
1028 (CA1:CA2:CA3:CA13:CA14:CA1580:200:589:139:10:10) 576 (CA1:CA2:CA3:CA4:CA5:CA6:CA7:CA8:CA9:CA10:CA11:CA12 77:26:44:11:363:18:11:7:7:4:4:4) 10 (CA16:CA17:CA18:CA19:CA20:CA21:CA222:2:2:1:1:1:1)
* Forestry and Forest Products Research Institute (FFPRI). ** National Institute of Advanced Industrial Science and Technology (AIST). *** The cultural characters of each actinomycete type were observed after one week of cultivation on ISP2 medium at 289C. The `Guide to Color Standard'10) was used to identify mycelium colors. The characters were shown in the order of character of aerial mycelium, color of aerial mycelium, color of substrate mycelium and soluble pigment. CA1: abundant, brownish gray, brownish black and yellow, CA2: abundant, brownish gray, brownish black and , CA3: abundant, gray, yellowish brown and brown, CA4: thin, light brown, pale ywllowish brown and , CA7: thin, brownish white, brownish white and , CA9: abundant, brownish black, black and , CA12: thin, yellowish gray, yellowish gray and , CA13: thick, pale orange, brown and brown, CA16: thick, pale brown, dull yellow and , CA17: thin, white, yellowish brown and , CA18: abundant, light gray, pale yellowish brown and , CA19: thick, pale orange, yellowish brown and , CA22: thin, pale brown, dull yellow and . In the case of the types unrecognized aerial mycelium, character of colony and color of substrate mycelium are shown. CA5: at and grayish yellow brown, CA6: wrinkled and brownish white, CA8: at and pale yellowish brown, CA10: at and yellowish gray, CA11: at and pale yellow, CA14: at and yellowish brown, CA15: at and dull yellow orange, CA20: at and brownish black, CA21: at and dark yellowish brown.
was analyzed by the dideoxy chain termination method with a BigDye terminator cycle sequencing kit (PE Applied Biosystems) and an automated DNA sequencer (ABI model 377, PE Applied Biosystems). Sequence alignments were done with the Clustal X multiple sequence alignment program.11) Ambiguous ly and incorrectly aligned positions were aligned manually or excepted from the analysis using the SeqPup sequence editor.12) Phylogenetic analysis was accomplished with the PHYLIP programpackage.13) Phylogenetic trees were constructed by using the neighborjoining distance matrix method 14) with 100 bootstrap replicates. The growth abilities of 4 actinomycete strains isolated from C. formosanus guts on plates of lignin and celluloserelated media were evaluated by cultivating for 1 week and examining the growth, both with the naked eye and under a stereoscopic microscope. Isolation of microorganisms from C. formosanus
collected from Iriomotejima was done on each of the media described above. The ratios of lamentous actinomycete colonies to other bacterial colonies that appeared on the celluloserelated Avicel and cellulose powder media were 0.027 and 0.006, respectively. On the other hand, on the ligninrelated dealkaline lignin and HVA media, the ratios of lamentous actinomy cete colonies to other bacterial colonies were 0.401 and 0.265, respectively. Moreover, very high ratios of lamentous actinomycetes were isolated on the defatted wood powder medium reported in this paper: the ratios were 38.38 and 9.025 for the termites taken from Iriomotejima and Yakushima, respectively. When the ligninrelated media were used, there was a high ratio of lamentous actinomy cete colonies to other bacterial colonies. Microorganisms from the guts of various species of termites were isolated on defatted wood powder medium (Table 2). The cultural characters of each of the actinomycete types were shown at the footnote of
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Y. WATANABE et al.
Table 2. From C. formosanus collected from Yakushima and Iriomotejima, we isolated many colonies of lamentous actinomycetes and a few colonies of other bacteria (although comprising only a limited number of types). On the other hand, no lamentous actinomycetes were isolated from C. formosanus kept at FFPRI (Forestry and Forest Products Research Institute); instead, many lamen tous fungi were isolated. The number of colonies and the actinomycete types on cultural characteristics iso lated from R. speratus collected from Yonagunijima were very similar to those of C. formosanus collected from Yakushima and Iriomotejima. On the other hand, from R. speratus in Tsukuba, only a small number of colonies appeared on the defatted wood powder medium. Some of the actinomycete types based on cultural characters isolated from N. koshunensis from Iriomotejima overlapped with those from C. formosanus and R. speratus collected from the islands in the southwest of Japan, but some of the types were recognized as unique to N. koshunensis. In case of H. japonica from Yakushi ma, some of the isolated actinomycete types over lapped with those found in other termite species, but most were unique to H. japonica. O. formosanus is a higher order termite which does not have protozoa in the gut. Being dierent from the other species of ter mites studied, the number of lamentous actinomy cete colonies isolated per individual termite was small, but the actinomycete types based on cultural characters were varied and nearly all were dierent from the actinomycete types isolated from the other lower order termites. These results show that the microorganism ora in the guts of termites mainly depends on the areas which the termites inhabit. In termites from the same area, microorganism ora in the gut depends on taxonomic dierences between termites. Identication by 16S rDNA analysis was done on actinomycete types CA1 and CA2, which were isolat ed largely from C. formosanus and R. speratus guts. The phylogenetic positions of types CA1 and CA2 are shown in Fig. 2 by using the neighborjoining distance matrix method.14) The actinomycete types CA1 and CA2 were the same species, belonging to the genus Streptomyces. The actinomycete types CA1 and CA2 grow abundantly on ISP2 medium, the type CA1 produces a soluble yellow pigment and the type CA2 doesn't (Table 2). The growth ability of the 4 actinomycete types isolated from C. formosanus was studied on lignin and celluloserelated media (Table 3). Isolates of types CA1 and CA2 grew relatively well on lignin related media. On the other hand, types CA3 and CA4 did not grow satisfactorily on ligninrelated media, but grew well on the celluloserelated media. Type CA3 grew especially well on the Avicel plate.
Fig. 2. Phylogenetic Positon of the Actinomycete Type CA1 Iso lated from Termite Guts. The sequence of type CA2 was identical with type CA1. The tree topology was tested by 100 repetitions of bootstrap analysis. The numbers followed by species name are accession numbers of the database (Genbank). The scale bars indicate 10z estimated sequence divergence.
Table 3. Growth on Lignin and Celluloserelated Media of Ac tinomycetes Isolated from Coptotermes formosanus Guts Medium
Actinomycete type CA1
CA2
CA3
CA4
Carboxymethyl cellulose (CMC) Avicel Cellulose powder
{ { {
{{ { {
{ {{ {
{ { {
0.2z Dealkaline lignin 0.5z Dealkaline lignin
{ }
{{ }
} }
} }
Cultivated for one week. {{: Good growth on the plate. {: Growth can be recognized with the naked eye. }: Growth can be recognized under a stereoscopic microscope.
Acknowledgments We gratefully acknowledge the nancial support for this study provided by the Bioconsortia Program, New Energy and Industrial Technology Development Organization (NEDO) as a part of the project ``Development of Technology of Bioconsortia and Utilization of Biological Resources'' conducted by the National Institute of Advanced Industrial Science and Technology.
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