Copyright 2010 The Society for Actinomycetes Japan VOL. 24, NO. 1
Actinomycetologica (2010) 24:12–17
Isolation and Characterization of Marine and Terrestrial Actinomycetes Using a Medium Supplemented with NaCl Chiaki Imada1 , Syunpei Masuda1 , Takeshi Kobayashi1 , Naoko Hamada-Sato1 , and Takuji Nakashima2 1
Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, 4-5-7, Konan, Minato-ku, Tokyo 108-8477, Japan 2 NITE Biotechnology Development Center (NBDC), Department of Biotechnology, National Institute of Technology and Evaluation (NITE), 2-5-8, Kazusakamatari, Kisarazu-shi, Chiba 292-0818, Japan. (At present: Kitasato Institute for Life Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.) (Received Mar. 24, 2010 / Accepted Apr. 26, 2010 / Published Jun. 25, 2010)
Actinomycetes isolated from marine sediments in neritic zone in the sea around Japan were compared with terrestrial isolates. Among actinomycetes that are macroscopically Streptomyces and Micromonospora, marine isolates of both genera were found to have higher NaCl tolerance than terrestrial ones. Around 37% of Streptomyces and 26% of Micromonospora that were isolated from the marine environment could tolerate up to 12% and 5% NaCl, respectively. However, no terrestrial isolates could tolerate NaCl at these concentrations. The results of the 16S rRNA gene sequence analysis of 10 strains with high NaCl tolerance among these marine isolates showed that they had a close phylogenetic relationship with terrestrial strains isolated previously. The isolation of actinomycetes from marine sediments and terrestrial soils in the presence or absence of 6% NaCl in media revealed that the selective pressure of NaCl demonstrated no clear difference between both isolates. Terrestrial strains isolated by using a medium supplemented with 6% NaCl exhibited a high frequency (95%) of NaCl tolerance up to 9%. The highly NaCl tolerant strains isolated from terrestrial soils were frequently found to produce antimicrobial substances in the presence of seawater. Almost 75% of strains tested showed antimicrobial activities against Bacillus subtilis PCI 219 and 40% against Candida albicans 3147. It was suggested that the frequency of microorganisms with antimicrobial properties was increased in the medium supplemented with seawater. ones. The marine environment is also expected to be an excellent sampling point due to microbial diversity in the seawater and sediment (Sugita et al., 1993). Recently, halophilic actinomycetes having a salt requirement similar to marine bacteria have reportedly been isolated from the marine environment. Novel metabolites, such as carcinostatic substances (Masky et al., 2003), aminoglycoside antibiotics (Hotta et al., 1980), cytotoxic substances (Kanoh et al., 2005) and other bioactive compounds (Imada 2009) were isolated from marine actinomycetes. Thus, the marine environment is expected to be a new screening target for finding novel bioactive compounds. The present study focused on the actinomycetes from marine sediments within the neritic zone in the sea around Japan. The characteristics and isolation methods for these marine actinomycetes were compared to terrestrial isolates and evaluated. Selective isolation of actinomycetes from marine sediments and terrestrial soils using a medium containing a high concentration of NaCl was performed. NaCl tolerance and production of antibiotic substances by these isolates was also examined.
INTRODUCTION Studies of actinomycetes to date have been mainly based on terrestrial strains. However, research into the beneficial microorganisms on the terrestrial environment has almost come to an end in recent years as different kinds of microorganisms basically produce the same compound, with respect to structure. In such situations, studies of terrestrial microbes in specific environments, such as a high-NaCl environment (Chen et al., 2004) and deserts (Takahashi et al., 1996) are increasing. Research concerning specific microbes that demonstrated a symbiotic relationship with plants (Igarashi 2004) is also increasing. However, studies on the isolation of microorganisms from marine environments have fallen behind due to the difficulties in collection of samples when compared to the terrestrial environment. The marine environment has several peculiar characteristics that are not seen in terrestrial areas, such as high hydrostatic pressure, high concentration of salts, and low concentration of organic matter. Microorganisms living in the marine environment are obviously expected to be different from terrestrial
Corresponding author and address: Chiaki Imada, 4-5-7, Konan, Minato-ku, Tokyo 108-8477, Japan. Tel: +81-3-5463-0404; Fax: +813-5463-0404; E-mail:
[email protected] 12
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branches from the resulting trees were evaluated by bootstrap analysis (Felsenstein 1985) of the neighborjoining method based on 1,000 replications.
MATERIALS AND METHODS Collection of marine sediment and terrestrial soil Marine sediment samples were collected using a piston core sediment sampler (depth > 100 m). A SmithMcIntyre sediment sampler (depth 10–100 m) and EkmanBirge bottom sediment sampler (depth < 10 m) were also employed. All the samples were stored at 20 C until required. Terrestrial soil samples were collected from a potato field in Yoshida-cho in Shizuoka Prefecture, leaf mold samples from forests and Hakone Onkei park in Hakone Yumoto in Kanagawa Prefecture. River sediment samples were collected from Arakawa river in the Tokyo metropolitan area.
Antibiotic production of the isolated strains Actinomycetes were cultivated in disposable plastic 24well plates (Sumitomo Bakelite Co. Ltd., Tokyo, Japan) for the screening of antibiotic substances. Cultivation was carried out in about 1.5 ml of ISP-2 liquid medium with or without synthetic seawater (Imada et al., 1998) at 27 C for 7 days on a minishaker shaking at 380 rpm (type MBSS500; B.E. Marubishi Co. Ltd, Tokyo, Japan). After cultivation, the culture was harvested by centrifugation at 9,000 g for 5 min and the supernatant was used to assay antibiotic activity by the cup assay method. The assay plates seeded with Bacillus subtilis PCI219 (Grampositive bacterium) using mycin assay agar, E. coli K-12 (Gram-negative bacterium) using nutrient agar and Candida albicans 3147 (yeast) using Sabouraud’s dextrose agar were used for the antibiotic assay. The antibiotic activity was determined after incubation at 27 C for 2 days.
Isolation of actinomycetes As an isolation medium, antibiotic substances such as nalidixic acid (20 mg/ml) and cycloheximide (50 mg/ml) were added to the autoclaved ISP-4 medium (Becton Dickinson and Co., Franklin Lakes, NJ, United States) after cooling to around 60 C, an isolation medium. Marine sediments or terrestrial soil samples (0.1 g each) were suspended in 0.1 ml saline solution and heated to 55 C for 30 min in order to eliminate bacterial growth. After treatment, the suspensions were spread on the medium and incubated at 27 C for 2 weeks until actinomycete colonies developed on the plates. For selective isolation of actinomycetes from marine sediment and terrestrial soil samples, isolation medium was prepared with the addition of various concentrations of NaCl.
RESULTS The number of actinomycetes strains obtained from marine and terrestrial areas was 241 and 143, respectively, in the present study. The isolates of marine origin were divided into two main categories, based on the isolated sea areas (inner bay and open ocean or deep-sea). Macroscopic observations were performed on all the isolates of both categories, and they were temporarily classified into two groups based on their morphologies. The large colonies and their color were white to gray in the early growth stage and then were covered with a powdery aerial mycelium in the later stages of growth. They were tentatively considered to be Streptomyces. On the other hand, the relatively smaller colonies were yellow-orange in the early stages of growth, changing to black or brown and forming a spore layer. They were tentatively considered to be Micromonospora. The sampling points within the sea area for isolation of actinomycetes are shown in Table 1.
Identification of actinomycetes with high NaCl tolerance by 16S rDNA analysis Ten actinomycetes encompassing four Streptomyces and six Micromonospora isolates that had an especially high NaCl tolerance were selected and subjected to identification by 16S rDNA sequence analysis. The almost complete 16S rDNA sequences of these strains were determined directly following PCR amplification. A fragment of the 16S rDNA (corresponding to positions 9–1510 in the Escherichia coli numbering system) was amplified by PCR. The reaction involved 35 cycles of denaturation at 98 C for 1 min, annealing at 55 C for 15 s and extension at 72 C for 1 min. PCR products were purified by using a Microcon Spin Column (Millipore Co., MA, United States). Both strands of purified fragments were sequenced directly using an ABI Prism Big Dye Terminator v3.1 kit (Applied Biosystems Japan, Tokyo, Japan) under conditions recommended by the manufacturer and using an automated sequence analyzer (model 3100; Applied Biosystems). The 16S rDNA sequences of isolates were compared with available 16S rDNA sequences from GenBank/DDBJ/EMBL using the BLAST program (Altschul et al., 1997). Phylogenetic trees were constructed by the neighbor-joining method (Saitou & Nei 1987) using the CLUSTAL W program (Thompson et al., 1994). The topological support of the internal
Streptomyces Sixty-two strains were isolated from the open ocean and deep sea such as Sagami Bay, South Pacific and the Japan sea, and 71 strains were isolated from inner bays such as Tokyo Bay, Otsuchi Bay in Iwate Prefecture, and Kagoshima Bay. Ninety-nine strains were isolated from leaf molds in forests and parks or river sediment around the Tokyo metropolitan area. Twenty-nine standard strains were obtained from the International Streptomyces Project (ISP). NaCl tolerance in these actinomycetes was examined in ISP-4 medium containing various concentrations of NaCl (0–15%) after incubation at 27 C for 4 weeks. 13
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Table 1. Sampling locations of actinomycetes in the marine environment. Station
Latitude (N) Longitude (E) Depth (m)
Open ocean and deep-sea Sagami Bay 34 59.930 South Pacific St.1 30 29.860 South Pacific St.2 33 08.250 Japan Sea St.1 35 12.600 Japan Sea St.2 36 30.100 East China Sea 31 50.730 Off-Boso 32 47.720
139 22.710 137 59.740 137 59.770 131 59.600 134 30.000 127 35.750 141 52.540
1,434 3,982 4,090 139 1,340 137 7,111
Inner bays Tokyo Bay St.1 Tokyo Bay St.2 Tokyo Bay St.3 Tokyo Bay St.4 Tokyo Bay St.5 Tokyo Bay St.6 Tokyo Bay St.7 Otsuchi Bay Kagoshima Bay
139 50.930 1395 7.860 139 59.130 139 55.190 139 52.110 139 48.050 139 47.100 141 58.930 130 40.950
3 8 11 21 22 22 3 60 193
35 37.620 35 39.250 35 36.130 35 31.310 35 25.650 35 30.790 35 31.490 39 21.350 31 39.490
Fig. 2. Comparison of NaCl tolerance between isolates from marine and terrestrial strains tentatively identified as Micromonospora.
Table 2. Actinomycetes isolates with high NaCl tolerance, isolated from the marine environment. Isolates
Sampling stations Sea area
SGB1-01 TKB1-01 TKB2-01 OTB1-01 OTB1-02 ECS1-02 SPO1-01 SPO3-01 SPO3-02 SPO3-03
Sagami Bay Tokyo Bay Tokyo Bay Otsuchi Bay Otsuchi Bay East China Sea South Pacific South Pacific South Pacific South Pacific
Depth (m) 1,434 20 20 65 65 137 3,982 4,090 4,090 4,090
NaCl Tentatively tolerance identified as (%) Streptomyces Streptomyces Streptomyces Streptomyces Micromonospora Micromonospora Micromonospora Micromonospora Micromonospora Micromonospora
12 12 12 12 5 5 5 5 5 5
2, respectively. The tolerance to NaCl of strains isolated from marine sediments was higher than terrestrial strains. It was apparent that all the isolates of marine origin had higher tolerance than terrestrial ones. In Streptomyces strains, 40% from inner bay and 33% from the open ocean were tolerant to NaCl concentrations up to 12%, but all terrestrial strains did not exhibit 12% NaCl tolerance. In Micromonospora strains, 18% from inner bay and 24% from the open ocean or deep sea were tolerant to NaCl at a concentration up to 5%, but none of the terrestrial or Streptomyces strains were tolerant to NaCl. Table 2 shows the location of sampling stations for 10 strains with especially high NaCl tolerance. Identification of actinomycetes having high NaCl tolerance by 16S rDNA analysis revealed that these strains had a close phylogenetic relationship with terrestrial strains (Fig. 3). Figure 4 shows the growth of the colonies of actinomycetes from marine sediment and terrestrial soil on the isolation medium
Fig. 1. Comparison of NaCl tolerance between isolates from marine and terrestrial strains tentatively identified as Streptomyces.
Micromonospora Seventy-nine strains were isolated from the open ocean and deep sea such as Sagami Bay, South Pacific Ocean, Japan Sea, Off-Boso and East China Sea, whereas 34 strains were isolated from inner bays such as Otsuchi Bay, Tokyo Bay, and Kagoshima Bay. Forty-four terrestrial strains were isolated from various places, as described above. The NaCl tolerance of Streptomyces and Micromonospora from each area of isolation is shown in Figures 1 and 14
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A
B
Fig. 3. Phylogenetic tree showing the position of 10 strains (A, 4 strains of Streptomyces; B, 6 strains of Micromonospora) with high NaCl tolerance.
marine sediments and terrestrial soils in the presence or absence of 6% NaCl in the medium revealed that the selective pressure of NaCl showed no clear difference between both isolates. This suggests that the selective pressure of NaCl is not always effective on marine isolates because they are living in seawater. On the other hand, 95% of terrestrial isolates from the isolation medium with 6% NaCl were tolerant to 9% NaCl. However, none of them could grow when the concentration of NaCl was 12%. Moreover, 75% and 40% of the strains isolated using the isolation medium with 6% NaCl exhibited antibiotic
containing various concentrations of NaCl. The results show that high concentrations of NaCl as a selective pressure affected the isolation of actinomycetes from both marine and terrestrial origins. Tables 3a and b show NaCl tolerance and antimicrobial activities of about 20 strains grown at the presence of 6% NaCl from marine sediments (Table 3a) and terrestrial soils (Table 3b). The actinomycetes isolated from the marine environments using the isolation medium containing NaCl did not exhibit apparent tolerance of NaCl or the production of antimicrobial substances. The isolation of actinomycetes from 15
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Table 3. a. NaCl tolerance and antimicrobial activities of strains isolated from marine sediments of Tokyo Bay by the addition of various concentrations of NaCl. b. NaCl tolerance and antimicrobial activities of the strains isolated from terrestrial soils by the addition of various concentrations of NaCl.
C
NaCl concentration (%) in isolation medium
a) marine
D
E
F
Fig. 4. (Color online) Photographs showing the growth of colonies on the selective isolation media containing various concentration of NaCl. Marine sediments (A, 0%; B, 3%; C, 6%), terrestrial soils (D, 0%; E, 3%; F, 6%).
0
3
6
NaCl tolerance 3% 6% 9% 12%
0 (0) 4 (21) 9 (47) 6 (32)
0 (0) 5 (24) 7 (33) 9 (43)
0 4 7 9
Total (%)
19 (100)
21 (100)
20 (100)
Antimicrobial activities against E. coli K-12 B. subtilis PCI219 C. albicans 3147
DW
DISCUSSION Actinomycetes are believed to be terrestrial in origin. It is well known that their NaCl tolerance is not as high when compared to actinomycetes of marine origin. Some terrestrial actinomycetes are reported to acquire salt tolerance gradually after being transported from a terrestrial to a marine environment by river water, rainfall or wind (Okami & Okazaki, 1975). An aureomycin-producing strain, which was identified as Streptomyces aureofaciens, is reported to have an extremely low NaCl tolerance (Okami & Okazaki, 1975). However, repeated subculturing of the strain in the medium containing seawater exhibited excellent growth due to adaptation to NaCl. The metabolites of the strain showed different UV-spectra from the parents, suggesting that NaCl not only increased the tolerance of the strain but altered the microbial metabolite (Imada et al., 2007). As shown in Figure 1, it is apparent that actinomycetes having a high NaCl tolerance (12%) in this study live in the marine environments. No terrestrial strains were able to grow at this concentration of NaCl. The actinomycetes that have high NaCl tolerance are expected to have a variety of metabolites and further study has revealed new findings. An attempt was made to isolate actinomycetes from marine sediment and terrestrial soil samples using medium containing 12% NaCl. However, it was hard to observe the actinomycete colonies on the medium even if they were incubated for a long period, suggesting that this is an extreme concentration for their growth. Therefore, selective isolation media containing 0– 6% NaCl were prepared and isolation of actinomycetes was
DW
SW
DW
SW
0 (0) 0 (0) 1 (5) 0 (0) 0 (0) 0 (0) 1 (5) 8 (42) 1 (5) 11 (52) 0 (0) 5 (25) 9 (47) 7 (37) 8 (38) 11 (52) 4 (20) 9 (45) NaCl concentration (%) in isolation medium
b) terrestrial
activity against B. subtilis PCI219 and C. albicans 3147, respectively, when they were cultivated in the medium containing seawater.
SW
(0) (20) (35) (45)
0
3
6
NaCl tolerance 3% 6% 9% 12%
5 (25) 11 (55) 4 (20) 0 (0)
1 (6) 6 (33) 11 (61) 0 (0)
0 (0) 1 (5) 19 (95) 0 (0)
Total (%)
20 (100)
18 (100)
20 (100)
Antimicrobial activities against E. coli K-12 B. subtilis PCI219 C. albicans 3147
DW
SW
DW
SW
0 (0) 2 (10) 1 (6) 3 (17) 7 (35) 9 (45) 5 (28) 8 (44) 3 (15) 6 (30) 3 (17) 3 (17)
DW
SW
0 (0) 0 (0) 2 (10) 15 (75) 8 (40) 8 (40)
No. of isolates (%) Production medium; DW, distilled water; SW, seawater.
performed. As shown in Table 3b, 95% of strains isolated with the medium containing 6% NaCl were tolerant to 9% NaCl, which is a relatively high concentration of NaCl for terrestrial actinomycetes. Actinomycetes are considered to be terrestrial microorganisms with low NaCl tolerance, therefore no attempt was made to isolate them using NaCl tolerance as selective pressure. In the present study, the percentage of antibioticproducing strains of isolates was approximately 20. The number of produced strains considerably increased compared to the ordinary isolation method. Thus, a selective isolation method using NaCl tolerance might be an effective way to find novel terrestrial actinomycetes from the environment. ACKNOWLEDGMENTS We are grateful to Dr. Yamamura for supplying the terrestrial Micromonospora strains used in the present study. 16
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substances produced by marine actinomycetes in the presence of seawater. Actinomycetologica 21, 27–31. Kanoh, K., Matsuo, Y., Adachi, K., Imagawa, H., Nishizawa, M. & Shizuri, Y. (2005). Mechercharmycins A and B, cytotoxic substances from marine-derived Thermoactinomyces sp. YM3-251. J. Antibiot. 58, 289–292. Masky, R. P., Li, F., Qin, S., Fiebig, H. H. & Laatsch, H. (2003). Chandrananilnycins production of novel anticancer antibiotics from a marine Actinomadura sp. isolate M048 by variation of medium composition and growth conditions. J. Antibiot. 56, 622–629. Okami, Y. & Okazaki, T. (1975). Actinomycetes tolerant to increased NaCl concentration and their metabolites. J. Ferment. Technol. 53, 833–840. Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425. Sugita, H., Sasanuma, S., Tanaka, K. & Deguchi, Y. (1993). Vitamin B12-producing ability of bacteria isolated from coastal water. Biosci. Biotechnol. Biochem. 57, 138–139. Takahashi, Y., Iwai, Y. & Omura, S. (1996). Rare actinomycetes s isolated from desert soils Actinomycetologica 10, 91–97. Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positionspecific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680.
REFERENCES Altschul, S. F., Madden, T. L., Scha¨ffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402. Chen, H. H., Li, W. J., Tang, S. K., Kroppenstedt, R. M., Stackebrandt, E., Xu, L. H. & Jiang, C. L. (2004). Corynebacterium halotolerans sp. nov., isolated from saline soil in the west of China. Int. J. Syst. Evol. Bacteriol. 54, 779– 782. Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791. Hotta, K., Yoshida, M., Hamada, M. & Okami, Y. (1980). Studies on new aminoglycoside antibiotics, istamycins, from an actinomycetes isolated from a marine environment. J. Antibiot. 33, 1515–1520. Igarashi, Y. (2004). Screening of novel bioactive compounds from plant-associated actinomycetes. Actinomycetologica 18, 63–66. Imada, C. (2009). Umino-biseibutsu-no-riyo. Seizando 83–87. Imada, C., Hotta, K. & Okami, Y. (1998). A novel marine Bacillus with multiple amino acid analog resistance and selenomethionine-dependent antibiotic productivity. J. Mar. Biotechnol. 6, 189–192. Imada, C., Koseki, N., Kamata, M., Kobayashi, T. & HamadaSato, N. (2007). Isolation and characterization of antibacterial
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