World J Microbiol Biotechnol (2013) 29:16771684 DOI 10.

1007/s11274-013-1330-4

ORIGINAL PAPER

An effective method based on wet-heat treatment for the selective isolation of Micromonospora from estuarine sediments
Takeshi Terahara Takeshi Kobayashi Chiaki Imada

Received: 16 December 2012 / Accepted: 19 March 2013 / Published online: 26 March 2013 Springer Science+Business Media Dordrecht 2013

Abstract Several methods for the isolation of Micromonospora from soil samples have been developed; however, it is unclear whether these methods are optimal for estuarine samples. In this study, we optimized the conditions of a wet-heat method for the selective isolation of Micromonospora from estuarine sediments. Sediments were collected from the Arakawa River (estuarine sediments) and Tokyo Bay (marine sediments). Sediment samples were wet-heated at 45, 55, or 65 C for 30 min and then incubated at 27 C for 3 weeks. After incubation, most of the actinomycete colonies were macroscopically determined to be of the genus Micromonospora or Streptomyces. In contrast to the treatment at 55 C, treatment at 65 C drastically reduced the number of Streptomyces colonies but increased the number of Micromonospora colonies from the estuarine sediments. This procedure allowed us to grow cultures that were composed of more than 90 % Micromonospora. In addition, treatment at 65 C did not affect the diversity of Micromonospora species compared with treatment at 55 C. These results indicate that the wetheat method, which involves pre-treating the sediment at 65 C for 30 min, is a very simple and effective method for the selective enrichment of a large number of diverse Micromonospora from estuarine sediments. Our results may lead to the isolation of new Micromonospora species,

which produce novel bioactive compounds, from different estuarine sediments. Keywords Actinobacteria Estuarine sediments Micromonospora Selective isolation Wet-heat method

Introduction Actinomycetes are abundant in soils and are considered benecial because they provide a wide variety of bioactive compounds. In particular, the genus Streptomyces is easy to cultivate and is mainly used for the commercial production of different antibiotics (Okami and Hotta 1988). Known bioactive compounds have often been discovered in many terrestrial Streptomyces isolated from different soils. The frequency of nding novel bioactive compounds has drastically decreased in recent years (Busti et al. 2006). Marine actinomycetes have recently been recognized as excellent sources of novel bioactive compounds (Dharmaraj 2010; Zotchev 2012), several of which are currently in clinical trials (Fenical and Jensen 2006). These discoveries may be associated with several environmental conditions unique to the marine environment, such as a high NaCl concentration, high hydrostatic pressure, low concentration of organic matter, and low temperature. It is hypothesized that marine actinomycetes may have unique characteristics that have never been found in terrestrial actinomycetes (Hodges et al. 2012). There is also growing interest in non-Streptomyces actinomycetes (rare actinomycetes), such as Salinospora (Mincer et al. 2002), that may be a unique source for novel bioactive compounds (Baltz 2006). Estuarine waters contain chemical gradients, such as salinity, nutrient concentration, and organic matter composition that

Electronic supplementary material The online version of this article (doi:10.1007/s11274-013-1330-4) contains supplementary material, which is available to authorized users.
T. Terahara (&) T. Kobayashi C. Imada Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, 4-5-7, Konan, Minato-ku, Tokyo 108-8477, Japan e-mail: terahara@kaiyodai.ac.jp

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result from mixing of freshwater and seawater. Many of these gradients are known to inuence the bacterial composition within estuaries (Barcina et al. 1997), forming unique microbial communities (Crump et al. 2004). In addition, Piza et al. (2004) reported that 16S rRNA clone libraries from estuarine sediments were related to sequences from non-cultured actinobacteria as well as sequences from unidentied bacteria detected in a wide range of environmental samples. Thus, estuarine sediments are attractive sources for the isolation of new groups of actinobacteria that produce novel bioactive compounds. The genus Micromonospora, one of the non-Streptomyces actinomycetes, is widely distributed in nature and is a well-known producer of antibiotics, such as gentamicin and netamicin (Berdy 2005). Micromonospora are often dominant members of the actinomycete communities, particularly in deep-sea marine sediments (Mincer et al. 2002); however, they are usually not dominant in estuarine sediments (Jensen et al. 1991). The use of selective enrichment is therefore crucial for the isolation of Micromonospora from soils and estuarine sediments. Selective enrichment of Micromonospora was rst accomplished by spreading soil samples over soil-extract agar plates (Nonomura and Ohara 1957). Since then, several methods of Micromonospora enrichment that involve the exploitation of the resistant properties of spores have been developed (Nonomura and Ohara 1969; Ivanitskaya et al. 1978; Goodfellow and Haynes 1984). Hayakawa et al. (1991) reported an effective method that involved pre-treating soil samples with 1.5 % phenol and using humic acid-vitamin (HV) agar. Qiu et al. (2008) described a simple and effective wet-heat method accomplished by pre-treating soil samples at 100 C for 1 h in a water bath and using ISP-3 agar medium. These previous studies have demonstrated effective methods of enriching Micromonospora from soil samples. In particular, heat treatment as a wet-heat method is a very simple method. Kawamoto (1992) reported that only 0.7 % of Streptomyces pactum spores and 0.2 % of S. griseus spores survived at 60 C for 10 min, whereas more than 50 % of Micromonospora spores survived at 60 C for 20 min. In addition, previous studies (Ensign 1982; Hoskisson et al. 2000) reported that heat-activation at mild temperatures (5070 C) stimulates spore germination, indicating that a large proportion of Micromonospora spores were constitutively dormant (Hoskisson et al. 2000). However, it is unclear whether the wet-heat method is suitable for estuarine samples, as estuarine sediments represent an environment that is very different from the environment associated with soil samples. To date, no studies have been undertaken to establish methods for the selective isolation of Micromonospora from estuarine sediments.

In the present study, we focus on examining optimal conditions of the wet-heat method for the selective isolation of Micromonospora from estuarine sediments. We evaluated the enrichment of Micromonospora in the examined conditions by determining the ratio of Micromonospora colonies to all actinomycete colonies. We also evaluated whether the heat-treatment conditions yielded diverse Micromonospora colonies. We sequenced the 16S rRNA gene amplied from isolated colonies and assessed the diversity of Micromonospora using a BLAST search, the resulting phylogenetic tree, and the Shannon-Wiener index (H0 ).

Materials and methods Collection of marine and estuarine sediment samples Estuarine sediment samples were collected from Arakawa River (Station 1 (St. 1) and St. 4) on 11 January 2011, 17 May 2011, and 30 May 2011 using an Ekman-Birge bottom sediment sampler (Rigo, Tokyo, Japan). Marine sediment sampling was also carried out in Tokyo Bay (St. 2 and St. 3). All of the samples were stored at -80 C prior to the isolation of actinomycetes. Several environmental factors, such as water temperature, salinity, and dissolved oxygen (DO), were measured using a multi-parameter probe (YSI model 556MPS, YSI/Nanotech Inc.) at each sampling station immediately after collection of surface water (Table 1). Isolation of actinomycetes ISP-3 and ISP-4 media containing cycloheximide (50 lg/ml), nystatin (50 lg/ml), and nalidixic acid (20 lg/ml) were used for the isolation of actinomycetes. The sediment samples (0.1 g each) were suspended in 1 ml of saline solution. Aliquots of 0.1 ml of the suspensions were diluted with 0.3 ml of a saline solution. To eliminate bacterial growth and selectively isolate Micromonospora, diluted suspensions were then incubated at 45, 55, or 65 C for 30 min because the viability of Micromonospora spores is known to be markedly reduced by heat pretreatment above 75 C (Suarez et al. 1980). After treatment, 0.1 ml of each suspension was spread over the medium in triplicate and incubated at 27 C for 3 weeks. After incubation, actinomycete colonies grown on the plates were macroscopically counted. The average of colony forming units (CFU/g of wet sediment) for each pretreated sediment sample was then used to estimate the ratio of Micromonospora colonies to all actinomycete colonies.

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World J Microbiol Biotechnol (2013) 29:16771684 Table 1 Sampling locations and environmental factors of seawater Sampling stations St.1 St.2 St.3 St.4 Longitude (E)/Latitude (N) 13950.740 /3536.910 14000.000 /3530.000 13952.110 /3525.650 13950.950 /3539.930 Sampling date 2011/1/11 2011/5/30 2011/1/11 2011/1/11 2011/5/17 2011/5/30 Depth (m) 2.8 2.8 15.9 21.5 21.1 3.6 Water temperature (C) 9.01 18.42 10.42 10.18 18.00 18.10 Salinity (ppt) 29.16 13.00 30.76 31.04 31.74 3.54

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DO (mg/L) 53.9 6.18 12.75 10.76 7.14 7.34

Identication of Micromonospora Members of the genus Micromonospora on ISP-4 medium were identied by 16S rRNA gene sequence analysis. A direct colony-PCR method was performed in which each reaction mixture (total of 25 ll) contained an individual actinomycete colony with 2X Buffer, 400 lM dNTPs, 0.2 lM each of universal primers (27f and 1492r), and 0.5 U of KOD FX DNA polymerase (Toyobo, Osaka, Japan). Amplication was performed under the following prole: 2 min at 94 C; 25 cycles of 10 s at 98 C, 30 s at 55 C, 90 s at 68 C; 4 min of nal extension at 68 C. The PCR products were veried by electrophoresis on 1.5 % (w/v) agarose gels, followed by 20 min of staining with ethidium bromide (10 ll l-1) and were puried with an Amicon Ultra-0.5 membrane lter (Millipore, Tokyo, Japan). DNA sequencing reactions were performed using the 27f primer and a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) according to the manufacturers protocol. The sequence data were collected on an ABI3130 Genetic Analyzer and subjected to a homology search using the Basic Local Alignment Search Tool (BLASTn) in the DNA Data Bank of Japan (DDBJ). The sequences from this study are available through the DDBJ under accession numbers AB748995-AB749048. Diversity analysis For diversity analysis of the Micromonospora colonies, the sequences from this study and 25 representative sequences of the genus Micromonospora were aligned using CLUSTAL W (Thompson et al. 1994). Subsequently, evolutionary distances were calculated using the program MEGA 4 (Tamura et al. 2007). A phylogenetic tree was then constructed by neighbor-joining analysis with distances based on the Kimura two-parameter method (Saitou and Nei 1987). Bootstrap replications (1,000) were performed using the MEGA program. In addition, the Shannon-Wiener diversity index (Shannon and Weaver 1963) was calculated according to the following equation:

H0

S P i 1

pi lnpi ; where pi is the ratio between the

number of colonies in a specic species and the total number of colonies, and S is the total number of species. The sequences from this study were classied into several species of the genus Micromonospora based on at least 97 % sequence similarity. This value has been reported as discriminating among bacterial species and was previously dened on the basis of DNADNA re-association values (Stackebrandt and Goebel 1994).

Results Isolation of actinomycetes Estuarine sediments (Arakawa River, St. 1 and St. 4) and marine sediments (Tokyo Bay, St. 2 and St. 3) were treated at 45, 55, or 65 C for 30 min. After incubation at 27 C for 3 weeks, the total number of actinomycete colonies from estuarine sediments ranged from 1.12 9 104 to 5.28 9 104 (CFU/g of wet sediment) on ISP-3 medium and from 2.28 9 104 to 3.12 9 104 (CFU/g) on ISP-4 medium (Table 2). For marine sediments, the total number of actinomycete colonies ranged from 0.6 9 104 to 1.84 9 104 (CFU/g) on ISP-3 medium and from 0.16 9 104 to 0.88 9 104 (CFU/g) on ISP-4 medium (Table 3). A comparison of the colony counts from estuarine sediments and marine sediments demonstrated that higher counts were recorded from estuarine sediments. In addition, as the treatment temperature increased, colony counts from the marine sediments tended to decrease, whereas colony counts from the estuarine sediments tended to increase. A similar correlation was observed in January and May. Most of the actinomycete colonies macroscopically resembled those of the genus Micromonospora or Streptomyces. The dominant genus depended on the water depth. In the marine sediments ([15 m depth), Micromonospora colonies were dominant in January and May under all treatment conditions (Table 3). On ISP-4 medium, Micromonospora

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Table 2 Effect of treatment temperature on the isolation of actinomycetes from estuarine sediments (9104 CFU/g of wet sediment) Sampling stations Date Treatment temperature (C) ISP-3 medium S* St.1 2011/1/11 No heat 45 55 65 2011/5/30 No heat 45 55 65 St.4 2011/5/30 No heat 45 55 65 2.76 1.96 1.44 0.2 0.64 0.6 0.44 0.04 1.56 1.44 1.12 0.12 M* 0.04 0.28 1.8 5.08 0.48 1.24 1.8 2.16 0.04 0.04 0.44 2.4 T* 2.8 2.24 3.24 5.28 1.12 1.84 2.24 2.2 1.6 1.48 1.56 2.52 ISP-4 medium S* 2.48 2.32 1.04 0.12 0.72 1.2 0.48 0.04 2.08 2.28 1.4 0.08 M* 0.28 0.2 1.28 2.68 2.4 1.88 2.48 2.48 0.2 0.2 1.32 2.56 T* 2.76 2.52 2.32 2.8 3.12 3.08 2.96 2.52 2.28 2.48 2.72 2.64

S* streptomyces colonies, M* micromonospora colonies, T* total actinomycete colonies Table 3 Effect of treatment temperature on the isolation of actinomycetes from marine sediments (9104 CFU/g of wet sediment) Sampling stations Date Treatment temperature (C) ISP-3 medium S* St.2 2011/1/11 No heat 45 55 65 St.3 2011/1/11 No heat 45 55 65 2011/5/17 No heat 45 55 65 0.04 0.04 0.04 0 0.04 0.12 0 0 0.12 0.08 0.08 0.12 M* 1.8 1.04 1.36 0.68 0.8 0.88 0.64 0.68 0.64 0.56 0.64 0.48 T* 1.84 1.08 1.4 0.68 0.84 1.0 0.64 0.68 0.76 0.64 0.72 0.6 ISP-4 medium S* 0.04 0 0.04 0 0.04 0.04 0 0 0.04 0.04 0.04 0 M* 0.64 0.32 0.6 0.36 0.32 0.4 0.32 0.16 0.84 0.56 0.56 0.36 T* 0.68 0.32 0.64 0.36 0.36 0.44 0.32 0.16 0.88 0.60 0.60 0.36

S* streptomyces colonies, M* micromonospora colonies, T* total actinomycete colonies

colonies composed more than 90 % of the actinomycete colonies (Fig. 1). Conversely, in the estuarine sediments (\5 m depth), Streptomyces colonies were dominant in January and May without wet-heat treatment (Table 2). However, the dominant genus shifted from Streptomyces to Micromonospora after heat-treatment conditions. Streptomyces colonies remained dominant after treatment at 45 C. Treatment at 55 or 65 C drastically reduced the number of Streptomyces colonies but increased the number of Micromonospora colonies (Table 2). On ISP-4 medium, the mean of Streptomyces colonies from estuarine sediments was 9.7 9 103 (CFU/g) after the 55 C treatment and 8.0 9 102 (CFU/g) after the 65 C treatment. Compared with no heat-

treatment, culturability of Streptomyces was reduced by approximately 40 % after the 55 C treatment and 95 % after the 65 C treatment. For Micromonospora colonies, the mean from estuarine sediments was 1.7 9 104 (CFU/g) after the 55 C treatment and 2.6 9 104 (CFU/g) after the 65 C treatment. Compared with no heat-treatment, culturability of Micromonospora was increased by approximately fourfold after the 55 C treatment and 7.8-fold after the 65 C treatment. Thus Micromonospora colonies were dominant and accounted for approximately 62 % of all actinomycete colonies after the 55 C treatment and more than 90 % after the 65 C treatment (Fig. 2). A similar tendency was observed on ISP-3 medium (data not shown). These results indicate that

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Fig. 1 The ratio of Micromonospora colonies to all actinomycete colonies in the sediment samples collected from Tokyo Bay (St. 2 and St. 3) using ISP-4 medium for colony growth

Fig. 2 The ratio of Micromonospora colonies to all actinomycete colonies in the sediment samples collected from Arakawa River (St. 1 and St. 4) using ISP-4 medium for colony growth

treatment at 65 C is a candidate for the selective isolation of Micromonospora from estuarine sediments. Diversity of Micromonospora colonies

selective isolation of a large number of diverse Micromonospora from estuarine sediments.

Discussion The macroscopically identied Micromonospora colonies isolated from St. 1 and St. 4 samples on ISP-4 medium were directly PCR amplied and sequenced using 16S rRNA gene universal primers. Based on the BLAST search in the current public databases (February, 2013), a total of 54 Micromonospora colonies were identied; sequences for which the homology score between the query sequence and the most similar sequence was less than 96 % were omitted. The sequence-identied Micromonospora species were classied into 3 types by differences in the isolation sources: (1) marine sediment samples (32 colonies), such as sandy beach, intertidal soil, and mangrove soil, (2) soil samples (11 colonies), and (3) other samples (11 colonies), such as plant and soft coral (Supplementary Table 1). Phylogenetic analysis showed that isolated Micromonospora species were diverse regardless of treatment at 55 or 65 C (Fig. 3); treatment at 65 C did not affect the diversity of isolated Micromonospora species compared with treatment at 55 C. In addition, the Shannon-Wiener diversity indices were calculated using the sequencing comparison results from the identied Micromonospora species. No signicant differences in the diversity of Micromonospora species were observed between treatment at 55 and 65 C (Table 4). Notably, the diversity of Micromonospora species tended to be inversely proportional to salinity at sampling stations. These results indicate that the wet-heat method, in which samples are treated at 65 C for 30 min, is a very simple and effective method for the In the present study, samples from estuarine sediments (St. 1 and St. 4 in Arakawa River, \5 m depth) and marine sediments (St. 2 and St. 3 in Tokyo Bay, [15 m depth) were pretreated at 45, 55, or 65 C for 30 min. The number of isolated actinomycete colonies was affected by water depth but not water temperature. Higher colony counts were recorded in estuarine sediment samples than in marine sediment samples (Tables 2, 3). A similar correlation was observed in January and May regardless of treatment conditions. The higher colony counts in samples from estuarine sediments appear to be due to the actinomycete origins, which are believed to be terrestrial sources (Goodfellow and Haynes 1984). Notably, as the treatment temperature increased, colony counts from marine sediments decreased, whereas colony counts from estuarine sediments increased (Tables 2, 3). The reason for this observed difference between estuarine and marine sediments is unclear, but one hypothesis is that dormant actinomycetes, which are starved and in a viable but non-culturable state due to chemical gradients that are tidally inuenced in the estuarine sediments, may become culturable due to the stimulation of spore germination following heat treatment. Water depth also affected which genus was most commonly cultured. Most of the isolated colonies were macroscopically determined to be of the genus Micromonospora or Streptomyces. Without treatment, Streptomyces colonies were the most common colony type in the estuarine sediments (\5 m depth), whereas Micromonospora

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1682 Fig. 3 Phylogenetic tree showing the relationships among the isolated species and 25 representative species of the genus Micromonospora based on 16S rRNA gene sequences. The tree was constructed by neighbor-joining analysis with distances based on the Kimura two-parameter method. Bootstrap percentages (1,000 replicates) above 50 % are shown at nodes. The isolated species were named after sampling station, month, treatment condition, and colony number

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colonies were the most common colony type in the marine sediments ([15 m depth) (Figs. 1, 2). These results recapitulated ndings from previous studies (Jensen et al. 1991; Takizawa et al. 1993; Bredholdt et al. 2007), suggesting that Streptomyces are the dominant actinomycetes in

terrestrial sources and then ow into estuaries, thus inuencing estuarine microbial community composition. Treatment at 55 or 65 C drastically reduced the number of Streptomyces colonies from estuarine sediments while increasing the number of Micromonospora colonies

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World J Microbiol Biotechnol (2013) 29:16771684 Table 4 ShannonWiener index (H0 ) of the culture results of estuarine sediments on ISP-4 medium

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Sampling stations St.1 St.1 St.4

Sampling date 2011/1/11 2011/5/30 2011/5/30

Salinity (ppt) 29.16 13.00 3.54

Treatment temperature 55 C 65 C 55 C 65 C 55 C 65 C

No. of identied colonies 6 9 8 11 11 9

ShannonWiener index (H0 ) 1.59 2.28 2.50 1.97 2.85 3.17

(Fig. 2). In particular, treatment at 65 C allowed us to grow cultures that were composed of more than 90 % Micromonospora colonies. This increase might be related to the resistant nature of Micromonospora spores. Previous electron-microscopy studies (Luedemann and Casmer 1973; Stevens 1975) revealed that the walls of Micromonospora spores have no inner multi-laminar coat, similar to that of Thermoactinomyces spores. Kawamoto (1992) reported that less than 1 % of Streptomyces spores survived at 60 C for 10 min, whereas more than 50 % of Micromonospora spores survived at 60 C for 20 min. These ndings suggest that Micromonospora spores are more heat-resistant than Streptomyces spores. We also evaluated whether heat-treatment enables the growth of diverse Micromonospora colonies from estuarine sediments. The phylogenetic tree and the Shannon-Wiener diversity indices revealed that treatment at 65 C did not signicantly affect the diversity of Micromonospora species compared with treatment at 55 C (Fig. 3; Table 4). This result may be due to the resistance of Micromonospora spores mentioned above. Notably, the salinity at the sampling stations affected the diversity of the Micromonospora colonies. The diversity indices tended to be inversely proportional to salinity in estuarine sediments (Table 4). This inverse relationship between salinity and diversity appears to be due to a low survival rate of Micronomospora in the sea because none of the known terrestrial Micronomospora are tolerant to 5 % NaCl (Imada et al. 2010). In addition, based on the BLAST search, the identied Micromonospora, which have the most similar sequences, have been isolated from various sources by other research groups, including marine sediment samples, soil samples, plant, and soft coral. These results are consistent with ndings from two research groups: Crump et al. (2004) reported that a unique microbial community was formed in estuarine samples. Piza et al. (2004) reported that 16S rRNA clone libraries from estuarine sediment were related to sequences detected from a wide range of environmental samples. In conclusion, we suggest that the wet-heat method in which samples are pre-treated at 65 C for 30 min

constitutes an ideal selection method for enriching Micromonospora from estuarine sediments. Our results demonstrate that the wet-heat method described above can aid in efforts to isolate new Micromonospora species from different estuarine sediments, which may lead to the identication of novel bioactive compounds.
Acknowledgments We thank the ofcers and crew members of the research and training vessel Hiyodori of Tokyo University of Marine Science and Technology for supporting sample collection for this study.

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