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Int. J. Mol. Sci. 2012, 13(5), 5917-5932; doi:10.3390/ijms13055917
Published: 16 May 2012
Abstract: The diversity and secondary metabolite potential of culturable actinomycetes associated with eight different marine sponges collected from the South China Sea and the Yellow sea were investigated. A total of 327 strains were isolated and 108 representative isolates were selected for phylogenetic analysis. Ten families and 13 genera of Actinomycetales were detected, among which five genera represent first records isolated from marine sponges. Oligotrophic medium M5 (water agar) proved to be efficient for selective isolation, and “Micromonospora–Streptomyces” was proposed as the major distribution group of sponge-associated actinomycetes from the China Seas. Ten isolates are likely to represent novel species. Sponge Hymeniacidon perleve was found to contain the highest genus diversity (seven genera) of actinomycetes. Housekeeping gene phylogenetic analyses of the isolates indicated one ubiquitous Micromonospora species, one unique Streptomyces species and one unique Verrucosispora phylogroup. Of the isolates, 27.5% displayed antimicrobial activity, and 91% contained polyketide synthase and/or nonribosomal peptide synthetase genes, indicating that these isolates had a high potential to produce secondary metabolites. The isolates from sponge Axinella sp. contained the highest presence of both antimicrobial activity and NRPS genes, while those from isolation medium DNBA showed the highest presence of antimicrobial activity and PKS I genes.
Marine sponges (Phylum Porifera) are multicellular invertebrate sessile filter-feeders that provide unique and favorable environmental conditions for microbial colonization and often harbor abundant and diverse microbes. Microbial communities associated with marine sponges are very complex, contributing up to 40% of the sponge biomass [1,2]. Marine sponge-associated bacterial communities include the following taxa: Acidobacteria, Actinobacteria, Bacteroidetes, Chlamydiae, Chloroflexi, Cyanobacteria, Deinococcus-Thermus, Firmicutes, Gemmatimonadetes, Nitrospira, Planctomycetes, Proteobacteria, Spirochaetes and Verrucomicrobia [3–7]. Among the bacterial associates, members of Actinobacteria are often sponge-specific [4,8] and have been identified as dominant producers of biologically active compounds [9–11]. There is evidence that the presence of biosynthesis genes encoding polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) in marine sponge-derived actinomycetes are useful indicators for the selection of strains to isolate new natural products .
Actinomycetes are widely distributed in marine sponges. At the time of writing, over 30 sponge genera had been reported to be hosts of actinomycetes, with ten genera having each been collected in different sea areas [4,5,7,8,13–18]. Among the nearly 10,000 sponge-derived microbial sequences submitted to public databases, about one-sixth belong to Actinobacteria , indicating that this is an important group among sponge-associated microorganisms. Actinomycetes abundance in marine sponges is variable but can make up over 20% of the total microorganisms in some marine sponges [20,21]. The study of marine-sponge-associated actinobacterial diversity involves both culture-dependent and culture-independent methods. In the past decade, a large number of marine sponge-derived actinomycetes have been identified using culture methods, spanning 26 genera [12,22–29]. The use of culture-independent methods has enabled the detection of an additional five genera of actinomycetes in marine sponges, as well as many unculturable novel actinobacterial taxa [21,30,31]. Although both of the above-mentioned methods have defects and bias, the culture-dependent method is still popular even in the “omics” age . This is partly because the isolates yielded from this method provide very useful phenotypic and genotypic information , such as physiological traits and biosynthetic potential, for further ecological investigation and bioprospecting.
Diverse sponges are found in the China Seas, with the South China Sea being estimated to contain nearly half of the marine sponge species in the world . The aim of this study was to investigate the diversity and biosynthetic potential of culturable actinomycetes associated with various sponges from the South China Sea and the Yellow Sea. To this end, several selective isolation media were used, and the isolates were subjected to phylogenetic analyses based on 16S rRNA and other housekeeping genes, and were tested for antimicrobial activity as well as the presence of secondary metabolite genes encoding polyketide synthases (PKS I and PKS II) and nonribosomal peptide synthetases (NRPSs).
2. Results and Discussion
2.1. Selective Isolation
A detailed description of the eight sponge samples that were processed is listed in Table 1. A quantitative analysis on the efficiency of the six media for actinomycete isolation is shown in Table 2. The water agar (M5) medium indicated the best isolation effect in terms of both selectivity and yield (percentage and number of actinomycete colonies). High selectivity values, but much lower yields, were recorded in the dilute nutrient broth agar (DNBA) and oatmeal agar (ISP 3). A good isolation effect, with relatively high selectivity and yield, was noted in water agar with sea salt (M5-S), while mannitol-peptone agars (SMP and SMP-S) were not effective in terms of isolating actinomycetes from sponges in the China Seas.
The quantitative data of this study indicated that oligotrophic media were suitable for the culture and isolation of marine sponge-associated actinomycetes. Medium M5, containing only agar and water, was the most suitable, in that it demonstrated the highest selectivity and yield, and the second-highest diversity of isolates. This contrasts with previous reports which showed that media containing appropriate nutrient were suitable for isolating actinomyctes from marine sponges [8,23,24,27–29] and is the first account of oligotrophic media being suitable for this purpose. Moreover, supplementing the medium with sea salt could not help the isolation because both the selectivity and yield of M5-S and SMP-S decreased. This is also supported by the fact that none of the isolates obligately depend on sea salt, as they all grew well on the purification medium yeast extract-malt extract agar (ISP 2) or starch-yeast extract-peptone agar (M1).
Most colonies fall into two main groups, based on colony morphology: the Micromonospora-like group (tiny, colored, solid colonies with unusual aerial hyphae) and the Streptomyces-like group (large colonies with aerial spore mass). The relative abundance between these two groups is 9:2, indicating that the Micromonospora-like actinomycetes were dominant among the sponges. A total of 327 isolates were purified from the isolation plates, based on sample ID, culture medium and strain morphology. Nearly half (46%) of the isolates were from sponges Axinyssa sp. WNGB3 (26%) and Xestospongia sp. QYP07 (20%), while the sponge Spongia sp. LCJ-1 yielded the smallest number of isolates (5%) (Table 1).
2.2. Phylogenetic Analysis and Diversity
According to morphological characteristics and the source sponge, 108 representative isolates were selected. The nearly full-length (> 1300 bp) 16S rRNA genes of representative isolates were sequenced and subjected to phylogenetic analysis. Results showed that these belonged to ten families and 13 genera—Bogoriellaceae (Georgenia), Geodermatophilaceae (Blastococcus), Micrococcaceae (Kocuria), Micromonosporaceae (Catenuloplanes, Micromonospora, Verrucosispora), Nocardiaceae (Gordonia), Nocardiopsaceae (Nocardiopsis), Pseudonocardineae (Pseudonocardia, Saccharomonospora), Streptomycetaceae (Streptomyces), Streptosporangiaceae (Nonomuraea), and Thermomonosporaceae (Actinomadura). The relative abundances of isolates from these genera were as follows: Micromonospora (56 isolates: 51.9%), Streptomyces (30 isolates: 27.8%), Verrucosispora (7 isolates: 6.5%), Nocardiopsis (3 isolates: 2.8%), Actinomadura (2 isolates: 1.9%), Catenuloplanes (2 isolates: 1.9%), Kocuria (2 isolates: 1.9%), and Gordonia, Georgenia, Blastococcus, Pseudonocardia, Nonomuraea, and Saccharomonospora (each with one isolate and a relative abundance of 0.9%). Around 26 genera of culturable actinomycetes isolated from marine sponges have so far been reported. Our study added five genera to this record: Actinomadura, Blastococcus, Catenuloplanes, Georgenia and Nonomuraea.
The isolates formed 35, 25 and 19 OTUs, defined by 16S rRNA gene sequence distances of 0.01, 0.02 and 0.03, respectively. The highest sequence similarities to recognized species with valid names were as follows: 100% (23 isolates), 99.0–99.9% (75 isolates) and <99.0% (10 isolates). The latter ten isolates (Table 3) may represent novel species of actinomycetes, among which strain FXJ6.011 has been established as the type strain of Micromonospora yangpuensis sp. nov. .
The majority (60.2%) of isolates fell into the family Micromonosporaceae and formed diversiform branches in the 16S rRNA gene tree within three genera (Figure 1a): Micromonospora, Verrucosispora and Catenuloplanes. The second-most diverse isolates fell into the genus Streptomyces, with a diversity of 98.2–100% 16S rRNA gene sequence similarities. Three large phylogenetic branches were circumscribed in the genera Micromonospora, Verrucosispora and Streptomyces, each containing isolates from different sponges (Figure 1a). Although members of Branch I were isolated from the South China Sea and the Yellow Sea sponges, they indicated a close relationship to each other, with ≤0.001 16S rRNA gene (>1300 bp) distances and ≤0.003 gyrB gene (1106 bp) distances (Figure 1b). According to quantitative evidence on the relationship of 16S rRNA sequence similarity to DNA-DNA re-association in actinomycetes , as well as evidence indicating that the gyrB-based genetic distance of 0.014 would correspond to 70% DNA homology among Micromonospora species , the 13 isolates of this branch were identified as a single Micromonospora species, which is widely distributed in sponges of the two China Seas. Branch III contained 12 closely-related Streptomyces isolates that were only detected in the South China Sea sponges, with ≤0.001 16S rRNA gene sequence distances. With the exception of isolate FXJ6.293, the other 11 isolates shared identical 16S rRNA and multi-locus (atpD-gyrB-recA-rpoB-trpB, >2500 bp) sequences (Figure 1c), and therefore belonged to the same species [38,39]. In contrast, Branch II contained diverse members of the genus Verrucosispora, with 16S rRNA gene distances of 0.005–0.021 and the gyrB gene distances of 0.029–0.085. Members of this branch have only been isolated from the South China Sea sponges.
The media associated with isolate diversity are shown in Figure 2a. Oatmeal agar (ISP 3) yielded the highest diversity of isolates, being associated with seven genera; water agar (M5) yielded the largest number of isolates, being associated with five genera, while water agar with sea salt (M5-S) yielded obviously fewer isolates of six genera; and dilute nutrient broth agar (DNBA) yielded isolates of only three genera. Sponge-associated isolate diversity is shown in Figure 2b. The most diverse group of actinomycetes was collected from sponge Hymeniacidon perleve MFDL, which contained seven genera, but albeit accounted for only 8% of the isolates. This was followed by the sponge host Xestospongia sp. SYM12, to which five actinomycetes genera were associated, and four genera were associated with each of the following three sponges: Xestospongia sp. QYP07, Axinyssa sp. WNGB3 and Dysidea sp. WNGB9. Sponges Axinella sp. XZNH and Reniochalina sp. SZDL each harbored three genera, and only two genera were associated with sponge Spongia sp. LCJ-1. Actinomycetes genera Micromonospora and Streptomyces were recovered on all of the media and from each sponge sample.
Selvin et al.  found that the “Micromonospora–Saccharomonospora–Streptomyces” group is a major culturable actinobacterial group in the marine sponge Dendrilla nigra. According to our investigation, Micromonospora spp. and Streptomyces spp. can be isolated from all of the eight sponges, but Saccharomonospora sp. was only isolated from the sponge Xestospongia sp QYP07. In seven out of eight sponges, the Micromonospora colony was dominant, and Streptomyces was the dominant genus in the sponge Axinella sp. XZNH and the sub-dominant genus in the other seven sponges. Taking these results into consideration, we propose that “Micromonospora–Streptomyces” is the major group of culturable marine sponge-associated actinomycetes in the China Seas.
The family Micromonosporaceae is widely distributed in the sea and five genera of this family have been recorded from marine environments: Asanoa, Micromonospora, Polymorphospora, Salinospora and Verrucosispora [40–43], of which Micromonospora, Salinospora and Verrucosispora can be isolated from marine sponges. In this study, the isolates of the family Micromonosporaceae were found to be dominant, in terms of quantity and diversity, in most marine sponges, and three genera of this family were isolated as well. However, we did not find the genus Salinospora; instead, we obtained Catenuloplanes. This is the first report of the genus Catenuloplanes from marine sponges.
2.3. Antimicrobial Activity and Gene Screening
Two hundred and forty-four isolates (including the 108 representative isolates) were tested for antimicrobial activity and the presence of PKS I, PKS II and NRPS genes (Table 4). Sixty-seven tested isolates (27.5%) displayed antimicrobial activity, belonging to the genera Streptomyces (34 isolates), Micromonospora (30 isolates), Actinomadura, Gordonia and Pseudonocardia (supplementary Table S1). Isolates associated with sponge Axinella sp. XZHN (mostly Streptomyces spp.) had the highest occurrence of antimicrobial activity (58.6%) compared with those of other sponge-associated isolates, notably the activity against Candidia albicans (31%). Of the tested isolates, 222 (91%) contained PKS/NRPS genes, and isolates associated with sponge Dysidea sp. WNGB9 showed the highest occurrence of PKS/NRPS genes (100%). The average detection rates of PKS I, PKS II and NRPS genes of the isolates were 54.9, 58.6, 60.2%, respectively, and the highest rates were detected in isolates from sponges Spongia sp. LCJ-1 (75.0%), Xestospongia sp. SYM12 (92.0%) and Axinella sp. XZHN (77.8%). Isolates from sponge Axinella sp. XZHN showed the highest presence of both antimicrobial activity and NRPS genes, indicating that they may possess good biosynthetic potential; while isolates from sponge Reniochalina sp. SZDL showed the lowest presence of both antimicrobial activity (16.1%) and PKS/NRPS genes (80.6%). It is also noticeable that isolates from dilute nutrient broth agar (DNBA) showed the highest presence of antimicrobial activity as well as PKS I gene (supplementary Table S2), despite the fact that this medium gave low yield and the genus diversity of the isolates.
Results from our study indicate that the actinomycetes from China Sea sponges have higher, or equal, percentages of antimicrobial activity as well as the presence of biosynthetic genes, in comparison with isolates derived from other marine environments [12,44,45]. It was shown that strains in which either PKS or NRPS genes were identified produced a significantly higher number of metabolites and exhibited a larger number of unidentified metabolites than other strains . Most organisms (91%) isolated in our study have the potential to produce secondary metabolites. Although only seven out of the 67 bioactive isolates were against E. coli, this is not frustrating because Gram-negative bacteria are generally more resistant to antibiotics due to their unique outer membrane, and we used only one medium (ISP 2 or M1) to cultivate the isolates for antimicrobial assay.
There is evidence that sponges have the capacity to “assemble” associated microorganisms, which would be useful for their survival [46–48]. Apart from rare pathogenic strains, actinomycetes are generally not harmful and can produce abundant bioactive metabolites [10,12,15,25,49], e.g., the widespread Micromonospora strains are not only useful in biomedicine, biocontrol and potentially in biofuels , but also closely related with nitrogen fixation in plant root nodules . This might be the major reason for the assembly of actinomycetes in marine sponges: for the protection and benefit of the sponge host. The identification of bioactive small molecules and gaining an understanding of their roles in the interactions between sponges and symbiotic actinomycetes will become a major focus of future studies.
3. Experimental Section
Six specimens of the marine sponges were collected from the South China Sea and two specimens from the Yellow Sea, by means of SCUBA diving to depths of up to 10 m. Samples were kept on ice in fresh seawater and later transported to the laboratory where they were stored at −20 °C.
3.2. Selective Isolation and Dereplication of Actinomycetes
To remove transient and loosely attached bacteria, each sponge sample was thoroughly washed at least 5 times with sterile water until clear, after which sponge material of 1 g was cut into pieces measuring approximately 1 cm3 and then homogenized in sterile mortars. Homogenates were heated in a water bath at 55 °C for 6 min, diluted in series, and plated in triplicate on agar plates. Based on the previous researches and pre-experiments, six media were used for isolation: oatmeal agar (ISP 3; DSMZ medium 609), mannitol-peptone agar (SMP) and mannitol-peptone agar with sea salt (SMP-S) , water agar (M5) and water agar with sea salt (M5-S) , and dilute nutrient broth agar (DNBA) . All of the media were supplemented with nalidixic acid and nystatin (each at 25 mg·L−1). The inoculated plates were incubated at 28 °C for 30–60 days. Colonies were counted and representative colonies from each sample were picked out and purified on yeast extract-malt extract agar (ISP 2; DSMZ medium 65) or starch-yeast extract-peptone agar (M1)  plates. The isolates were divided into different groups according to a number of morphological characteristics, including color of aerial and substrate mycelia, diffusible pigments, arrangement of hyphae and spore chains, and spore shape.
3.3. Antimicrobial Activity Screening
An agar diffusion assay was used for antimicrobial screening against indicator microorganisms, including bacteria Bacillus subtilis CGMCC 1.2428 (=DSM 347), Escherichia coli CGMCC 1.2385 (=DSM 1103) and Staphylococcus aureus subsp. aureus CGMCC 1.2386 (=DSM 1104), as well as yeast Candidia albicans CGMCC 2.538. Agar plugs that were fully covered with a mass of isolated strains were cut from agar plates (ISP 2 or M1 media) that were incubated at 28 °C for 14 days, and transferred to test plates containing individual indicator strains, after which they were incubated at either 28 °C or 37 °C for 12–24 h. Diameters of the inhibition zone were measured for the purpose of describing the antimicrobial activity.
3.4. DNA Extraction, 16S rRNA and Housekeeping Gene Amplification
Extraction of genomic DNA, PCR amplification and sequencing of the 16S rRNA gene were performed, following the methods described by Chun and Goodfellow . The methods used for PCR amplification of the gyrB (DNA gyrase B subunit) gene and sequencing of the PCR products in members of the family Micromonosporaceae are outlined in Garcia et al. . For Streptomyces strains, five housekeeping genes—atpD (ATP synthase F1, β-subunit), gyrB, recA (recombinase A), rpoB (RNA polymerase, β-subunit) and trpB (tryptophan synthase, β-subunit)—were amplified and sequenced according to the methods developed by Guo et al.  and Rong et al. .
3.5. Phylogenetic Analysis
The resultant 16S rRNA gene (>1300 bp) and gyrB gene (>1100 bp) sequences were compared with those deposited in the public databases using the NCBI BLAST program and were aligned with related sequences retrieved from the public databases using the Clustal W algorithm in MEGA version 5.0 . The gene sequence similarity values were calculated after pairwise alignment using MEGA version 5.0. The five housekeeping gene sequences (ca. 500 bp each) of the isolates were aligned and trimmed manually at the same position, prior to multi-locus sequence analysis, and concatenated by joining head-to-tail in-frame in the following order: atpD-gyrB-recA-rpoB-trpB (>2500 bp). Phylogenetic trees were constructed in MEGA version 5.0 using the neighbor-joining method . Evolutionary distance matrices were calculated with the Kimura 2-parameter model. The Bootstrap test  was used to evaluate the reliability of the inferred tree, based on 1000 replications.
3.6. Detection of NRPS and PKS Genes
To assess the genetic potential of the isolates for producing bioactive secondary metabolites, the amplification of genes encoding polyketide synthases (PKS I and PKS II) and non-ribosomal peptide synthetase (NRPS) from the isolates was carried out using degenerate primers recommended by Ayuso-Sacido & Genilloud  and Metsa-Ketela et al. . The PCR amplification reagents and programs were based on those of Rong et al. .
3.7. Nucleotide Sequence Accession Numbers
The 16S rRNA gene sequences of representative isolates were deposited in GenBank database under the following accession numbers: EU914133, EU914135, EU914137, GU002066, GU002068-GU002076, GU002079, GU002081-GU002088, GU002090-GU002100, GU002103, JF346429-JF346431, JF346433-JF346482 and JN182138-JN182158. GenBank accession numbers for the partial gyrB sequences of Micromonospora and Verrucosispora isolates were JN182159-JN182194, and those for the housekeeping genes of Streptomyces isolates were JQ258943-JQ259047.
This research demonstrates the rich diversity of culturable actinomycetes from marine sponges in the South China Sea and the Yellow sea, with different, and similar, population structures. Five genera represent first records associated with marine sponges, and ten isolates are likely to represent novel species. Oligotrophic medium seems to be efficient for selective isolation. Antimicrobial activity and PKS/NRPS gene screening indicated that, with “Micromonospora–Streptomyces” as the major group, the sponge-associated actinomycetes from the China Seas have high biosynthetic potential, and could serve as a good resource for the exploration of bioactive natural products.
We thank Qianqun Gu (Ocean University of China) and Wei Zhang (Dalian Institute of Chemical Physics, Chinese Academy of Sciences) for sponge sample collection. We are also grateful to Danheng Qiu and Fang Yang (Institute of Microbiology, Chinese Academy of Sciences) for their valuable assistance with isolation and statistics analysis.
This work was supported by the Knowledge Innovation Program of Chinese Academy of Sciences (grant No. KSCX2-EW-J-6/KSCX2-EW-G-12B), and by the National Hi-Tech Research and Development Program of China (grant No. 2007AA09Z420).
- Friedrich, A.B.; Fischer, I.; Proksch, P.; Hacker, J.; Hentschel, U. Temporal variation of the microbial community associated with the Mediterranean sponge Aplysina aerophoba. FEMS Microbiol. Ecol 2001, 38, 105–115.
- Taylor, M.W.; Radax, R.; Steger, D.; Wagner, M. Sponge-associated microorganisms: Evolution, ecology, and biotechnological potential. Microbiol. Mol. Biol. Rev 2007, 71, 295–347.
- Hardoim, C.C.; Costa, R.; Araujo, F.V.; Hajdu, E.; Peixoto, R.; Lins, U.; Rosado, A.S.; van Elsas, J.D. Diversity of bacteria in the marine sponge Aplysina fulva in Brazilian coastal waters. Appl. Environ. Microbiol 2009, 75, 3331–3343.
- Kamke, J.; Taylor, M.W.; Schmitt, S. Activity profiles for marine sponge-associated bacteria obtained by 16S rRNA vs 16S rRNA gene comparisons. ISME J 2010, 4, 498–508.
- Radwan, M.; Hanora, A.; Zan, J.; Mohamed, N.M.; Abo-Elmatty, D.M.; Abou-El-Ela, S.H.; Hill, R.T. Bacterial community analyses of two red sea sponges. Mar. Biotechnol. (N.Y.) 2010, 12, 350–360.
- Webster, N.S.; Wilson, K.J.; Blackall, L.L.; Hill, R.T. Phylogenetic diversity of bacteria associated with the marine sponge Rhopaloeides odorabile. Appl. Environ. Microbiol 2001, 67, 434–444.
- Zhu, P.; Li, Q.; Wang, G. Unique microbial signatures of the alien Hawaiian marine sponge Suberites zeteki. Microb. Ecol 2008, 55, 406–414.
- Selvin, J.; Gandhimathi, R.; Kiran, G.S.; Priya, S.S.; Ravji, T.R.; Hema, T.A. Culturable heterotrophic bacteria from the marine sponge Dendrilla nigra: Isolation and phylogenetic diversity of actinobacteria. Helgol. Mar. Res 2009, 63, 239–247.
- Lang, S.; Beil, W.; Tokuda, H.; Wicke, C.; Lurtz, V. Improved production of bioactive glucosylmannosyl-glycerolipid by sponge-associated Microbacterium species. Mar. Biotechnol. (N.Y.) 2004, 6, 152–156.
- Thomas, T.R.A.; Kavlekar, D.P.; LokaBharathi, P.A. Marine drugs from sponge-microbe association—A review. Mar. Drugs 2010, 8, 1417–1468.
- Molinski, T.F.; Dalisay, D.S.; Lievens, S.L.; Saludes, J.P. Drug development from marine natural products. Nat. Rev. Drug Discov 2008, 8, 69–85.
- Schneemann, I.; Nagel, K.; Kajahn, I.; Labes, A.; Wiese, J.; Imhoff, J.F. Comprehensive investigation of marine actinobacteria associated with the sponge Halichondria panicea. Appl. Environ. Microb 2010, 76, 3702–3714.
- Cassler, M.; Peterson, C.L.; Ledger, A.; Pomponi, S.A.; Wright, A.E.; Winegar, R.; McCarthy, P.J.; Lopez, J.V. Use of real-time qPCR to quantify members of the unculturable heterotrophic bacterial community in a deep sea marine sponge, Vetulina sp. Microb. Ecol 2008, 55, 384–394.
- Isaacs, L.T.; Kan, J.; Nguyen, L.; Videau, P.; Anderson, M.A.; Wright, T.L.; Hill, R.T. Comparison of the bacterial communities of wild and captive sponge Clathria prolifera from the Chesapeake Bay. Mar. Biotechnol. (N.Y.) 2009, 11, 758–770.
- Izumikawa, M.; Khan, S.T.; Takagi, M.; Shin-ya, K. Sponge-derived Streptomyces producing isoprenoids via the mevalonate pathway. J. Nat. Prod 2010, 73, 208–212.
- Kennedy, J.; Baker, P.; Piper, C.; Cotter, P.D.; Walsh, M.; Mooij, M.J.; Bourke, M.B.; Rea, M.C.; O’Connor, P.M.; Ross, R.P.; et al. Isolation and analysis of bacteria with antimicrobial activities from the marine sponge Haliclona simulans collected from Irish waters. Mar. Biotechnol. (N.Y.) 2009, 11, 384–396.
- Sharp, K.H.; Eam, B.; Faulkner, D.J.; Haygood, M.G. Vertical transmission of diverse microbes in the tropical sponge Corticium sp. Appl. Environ. Microbiol 2007, 73, 622–629.
- Webster, N.S.; Cobb, R.E.; Negri, A.P. Temperature thresholds for bacterial symbiosis with a sponge. ISME J 2008, 2, 830–842.
- Webster, N.S.; Taylor, M.W. Marine sponges and their microbial symbionts: Love and other relationships. Environ. Microbiol 2011, 14, 335–346.
- Gandhimathi, R.; Arunkumar, M.; Selvin, J.; Thangavelu, T.; Sivaramakrishnan, S.; Kiran, G.S.; Shanmughapriya, S.; Natarajaseenivasan, K. Antimicrobial potential of sponge associated marine actinomycetes. J. Mycol. Med 2008, 18, 16–22.
- Montalvo, N.F.; Mohamed, N.M.; Enticknap, J.J.; Hill, R.T. Novel actinobacteria from marine sponges. Anton. Leeuwenhoek 2005, 87, 29–36.
- Abdelmohsen, U.R.; Pimentel-Elardo, S.M.; Hanora, A.; Radwan, M.; Abou-El-Ela, S.H.; Ahmed, S.; Hentschel, U. Isolation, phylogenetic analysis and anti-infective activity screening of marine sponge-associated actinomycetes. Mar. Drugs 2010, 8, 399–412.
- Jiang, S.M.; Li, X.; Zhang, L.; Sun, W.; Dai, S.K.; Xie, L.W.; Liu, Y.H.; Lee, K.J. Culturable actinobacteria isolated from marine sponge Iotrochota sp. Mar. Biol 2008, 153, 945–952.
- Jiang, S.; Sun, W.; Chen, M.; Dai, S.; Zhang, L.; Liu, Y.; Lee, K.J.; Li, X. Diversity of culturable actinobacteria isolated from marine sponge Haliclona sp. Anton. Leeuwenhoek 2007, 92, 405–416.
- Kim, T.K.; Fuerst, J.A. Diversity of polyketide synthase genes from bacteria associated with the marine sponge Pseudoceratina clavata: Culture-dependent and culture-independent approaches. Environ. Microbiol 2006, 8, 1460–1470.
- Lafi, F.F.; Garson, M.J.; Fuerst, J.A. Culturable bacterial symbionts isolated from two distinct sponge species (Pseudoceratina clavata and Rhabdastrella globostellata) from the Great Barrier Reef display similar phylogenetic diversity. Microb. Ecol 2005, 50, 213–220.
- Sun, W.; Dai, S.K.; Jiang, S.M.; Wang, G.H.; Liu, G.H.; Wu, H.B.; Li, X. Culture-dependent and culture-independent diversity of Actinobacteria associated with the marine sponge Hymeniacidon perleve from the South China Sea. Anton. Leeuwenhoek 2010, 98, 65–75.
- Zhang, H.; Lee, Y.K.; Zhang, W.; Lee, H.K. Culturable actinobacteria from the marine sponge Hymeniacidon perleve: Isolation and phylogenetic diversity by 16S rRNA gene-RFLP analysis. Anton. Leeuwenhoek 2006, 90, 159–169.
- Zhang, H.; Zhang, W.; Jin, Y.; Jin, M.; Yu, X. A comparative study on the phylogenetic diversity of culturable actinobacteria isolated from five marine sponge species. Anton. Leeuwenhoek 2008, 93, 241–248.
- Gerce, B.; Schwartz, T.; Voigt, M.; Ruhle, S.; Kirchen, S.; Putz, A.; Proksch, P.; Obst, U.; Syldatk, C.; Hausmann, R. Morphological, bacterial, and secondary metabolite changes of Aplysina aerophoba upon long-term maintenance under artificial conditions. Microb. Ecol 2009, 58, 865–878.
- Xin, Y.; Huang, J.; Deng, M.; Zhang, W. Culture-independent nested PCR method reveals high diversity of actinobacteria associated with the marine sponges Hymeniacidon perleve and Sponge sp. Anton. Leeuwenhoek 2008, 94, 533–542.
- Giovannoni, S.; Stingl, U. The importance of culturing bacterioplankton in the “omics” age. Nat. Rev. Microbiol 2007, 5, 820–826.
- Galkiewicz, J.P.; Pratte, Z.A.; Gray, M.A.; Kellogg, C.A. Characterization of culturable bacteria isolated from the cold-water coral Lophelia pertusa. FEMS Microbiol. Ecol 2011, 77, 333–346.
- Hooper, J.; Kennedy, J.; van Soest, R. Annotated checklist of sponges (Porifera) of the South China Sea region. Raffles Bull. Zool 2000, 125–207.
- Zhang, L.M.; Xi, L.J.; Ruan, J.S.; Huang, Y. Micromonospora yangpuensis sp. nov., isolated from a sponge in South China Sea. Int. J. Syst. Evol. Microbiol 2011, 62, 272–278.
- Stackebrandt, E.; Ebers, J. Taxonomic parameters revisited: Tarnished gold standards. Microbiol. Today 2006, 33, 152–155.
- Kasai, H.; Tamura, T.; Harayama, S. Intrageneric relationships among Micromonospora species deduced from gyrB-based phylogeny and DNA relatedness. Int. J. Syst. Evol. Microbiol 2000, 50, 127–134.
- Rong, X.Y.; Huang, Y. Taxonomic evaluation of the Streptomyces griseus clade using multilocus sequence analysis and DNA-DNA hybridization, with proposal to combine 29 species and three subspecies as 11 genomic species. Int. J. Syst. Evol. Microbiol 2010, 60, 696–703.
- Rong, X.Y.; Huang, Y. Taxonomic evaluation of the Streptomyces hygroscopicus clade using multilocus sequence analysis and DNA-DNA hybridization, validating the MLSA scheme for systematics of the whole genus. Syst. Appl. Microbiol 2012, 35, 7–18.
- Dai, H.Q.; Wang, J.A.; Xin, Y.H.; Pei, G.; Tang, S.K.; Ren, B.A.; Ward, A.; Ruan, J.S.; Li, W.J.; Zhang, L.X. Verrucosispora sediminis sp nov., a cyclodipeptide-producing actinomycete from deep-sea sediment. Int. J. Syst. Evol. Microbiol 2010, 60, 1807–1812.
- Maldonado, L.A.; Fenical, W.; Jensen, P.R.; Kauffman, C.A.; Mincer, T.J.; Ward, A.C.; Bull, A.T.; Goodfellow, M. Salinispora arenicola gen. nov., sp nov and Salinispora tropica sp nov., obligate marine actinomycetes belonging to the family Micromonosporaceae. Int. J. Syst. Evol. Microbiol 2005, 55, 1759–1766.
- Tamura, T.; Sakane, T. Asanoa iriomotensis sp nov., isolated from mangrove soil. Int. J. Syst. Evol. Microbiol 2005, 55, 725–727.
- Tamura, T.; Hatano, K.; Suzuki, K. A new genus of the family Micromonosporaceae, Polymorphospora gen. nov., with description of Polymorphospora rubra sp. nov. Int. J. Syst. Evol. Microbiol 2006, 56, 1959–1964.
- Bredholt, H.; Fjaervik, E.; Johnsen, G.; Zotchev, S.B. Actinomycetes from Sediments in the Trondheim Fjord, Norway: Diversity and biological activity. Mar. Drugs 2008, 6, 12–24.
- Penesyan, A.; Marshall-Jones, Z.; Holmstrom, C.; Kjelleberg, S.; Egan, S. Antimicrobial activity observed among cultured marine epiphytic bacteria reflects their potential as a source of new drugs. FEMS Microbiol. Ecol 2009, 69, 113–124.
- Sacristan-Soriano, O.; Banaigs, B.; Casamayor, E.O.; Becerro, M.A. Exploring the links between natural products and bacterial assemblages in the sponge Aplysina aerophoba. Appl. Environ. Microbiol 2011, 77, 862–870.
- Luter, H.M.; Whalan, S.; Webster, N.S. Exploring the role of microorganisms in the disease-like syndrome affecting the sponge Ianthella basta. Appl. Environ. Microbiol 2010, 76, 5736–5744.
- Thomas, T.; Rusch, D.; DeMaere, M.Z.; Yung, P.Y.; Lewis, M.; Halpern, A.; Heidelberg, K.B.; Egan, S.; Steinberg, P.D.; Kjelleberg, S. Functional genomic signatures of sponge bacteria reveal unique and shared features of symbiosis. ISME J 2010, 4, 1557–1567.
- Kim, T.K.; Hewavitharana, A.K.; Shaw, P.N.; Fuerst, J.A. Discovery of a new source of rifamycin antibiotics in marine sponge actinobacteria by phylogenetic prediction. Appl. Environ. Microbiol 2006, 72, 2118–2125.
- Hirsch, A.M.; Valdes, M. Micromonospora: An important microbe for biomedicine and potentially for biocontrol and biofuels. Soil Biol. Biochem 2010, 42, 536–542.
- Trujillo, M.E.; Alonso-Vega, P.; Rodriguez, R.; Carro, L.; Cerda, E.; Alonso, P.; Martinez-Molina, E. The genus Micromonospora is widespread in legume root nodules: The example of Lupinus angustifolius. ISME J 2010, 4, 1265–1281.
- Jensen, P.R.; Gontang, E.; Mafnas, C.; Mincer, T.J.; Fenical, W. Culturable marine actinomycete diversity from tropical Pacific Ocean sediments. Environ. Microbiol 2005, 7, 1039–1048.
- Mincer, T.J.; Fenical, W.; Jensen, P.R. Culture-dependent and culture-independent diversity within the obligate marine actinomycete genus Salinispora. Appl. Environ. Microbiol 2005, 71, 7019–7028.
- Janssen, P.H.; Yates, P.S.; Grinton, B.E.; Taylor, P.M.; Sait, M. Improved culturability of soil bacteria and isolation in pure culture of novel members of the divisions Acidobacteria, Actinobacteria, Proteobacteria, and Verrucomicrobia. Appl. Environ. Microbiol 2002, 68, 2391–2396.
- Chun, J.; Goodfellow, M. A phylogenetic analysis of the genus Nocardia with 16S rRNA gene sequences. Int. J. Syst. Bacteriol 1995, 45, 240–245.
- Garcia, L.C.; Martinez-Molina, E.; Trujillo, M.E. Micromonospora pisi sp nov., isolated from root nodules of Pisum sativum. Int. J. Syst. Evol. Microbiol 2010, 60, 331–337.
- Guo, Y.; Zheng, W.; Rong, X.; Huang, Y. A multilocus phylogeny of the Streptomyces griseus 16S rRNA gene clade: Use of multilocus sequence analysis for streptomycete systematics. Int. J. Syst. Evol. Microbiol 2008, 58, 149–159.
- Rong, X.Y.; Guo, Y.P.; Huang, Y. Proposal to reclassify the Streptomyces albidoflavus clade on the basis of multilocus sequence analysis and DNA-DNA hybridization, and taxonomic elucidation of Streptomyces griseus subsp. solvifaciens. Syst. Appl. Microbiol 2009, 32, 314–322.
- Tamura, K.; Peterson, D.; Peterson, N.; Stecher, G.; Nei, M.; Kumar, S. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol 2011, 28, 2731–2739.
- Saitou, N.; Nei, M. The neighbor-joining method—A new method for reconstructing phylogenetic trees. Mol. Biol. Evol 1987, 4, 406–425.
- Felsenstein, J. Confidence-limits on phylogenies—An approach using the bootstrap. Evolution 1985, 39, 783–791.
- Ayuso-Sacido, A.; Genilloud, O. New PCR primers for the screening of NRPS and PKS-I systems in actinomycetes: Detection and distribution of these biosynthetic gene sequences in major taxonomic groups. Microb. Ecol 2005, 49, 10–24.
- Metsa-Ketela, M.; Salo, V.; Halo, L.; Hautala, A.; Hakala, J.; Mantsala, P.; Ylihonko, K. An efficient approach for screening minimal PKS genes from Streptomyces. FEMS Microbiol. Lett 1999, 180, 1–6.
|Table 1. Detailed accounts of the sponge samples collected from the Yellow Sea and the South China Sea.|
|Sample ID||Sponge Species||Location||Collection Time||No. of Isolates (%)|
|MFDL||Hymeniacidon perleve||YS, N38°51.497 E121°32.357||2009.03||26 (8)|
|SZDL||Reniochalina sp.||YS, N38°51.497 E121°32.357||2009.03||35 (11)|
|LCJ-1||Spongia sp.||SCS, N19°55.269 E109°29.118||2006.04||15 (5)|
|QYP07||Xestospongia sp.||SCS, N19°39.925 E109°04.678||2006.04||65 (20)|
|SYM12||Xestospongia sp.||SCS, N18°19.478 E108°57.229||2006.03||44 (13)|
|WNGB3||Axinyssa sp.||SCS, N18°53.068 E110°31.314||2006.04||85 (26)|
|WNGB9||Dysidea sp.||SCS, N18°53.068 E110°31.314||2006.04||27 (8)|
|XZHN||Axinella sp.||SCS, N16°83 E121°33||2009.03||30 (9)|
YS, Yellow Sea; SCS, South China Sea.
|Table 2. Colony numbers of actinomycetes and non-actinomycetes recorded on six different selective media seeded with all eight samples.|
|Medium||Total No. (%) of Actinomycetes||Total No. (%) of Other Microbes||No. of Actinomycetes per Plate|
|Dilute nutrient broth agar (DNBA)||427 (96)||17 (4)||9|
|Oatmeal agar (ISP 3)||126 (93)||9 (7)||3|
|Water agar (M5)||2076 (96)||83 (4)||44|
|Water agar with sea salt (M5-S)||1318 (90)||152 (10)||28|
|Mannitol-peptone agar (SMP)||1712 (45)||2105 (55)||36|
|Mannitol-peptone agar with sea salt (SMP-S)||690 (10)||6189 (90)||15|
|Table 3. The ten isolates having <99% 16S rRNA gene sequence similarities with the closest species.|
|Isolate||GenBank No.||Closest Species||Type strain’s GenBank No.||Similarity (%)||Isolation Medium||Source Sponge|
|FXJ6.011||GU002071||Micromonospora chaiyaphunensis||AB196710||98.7||SMP-S||Xestospongia sp. QYP07|
|FXJ6.013||GU002073||Verrucosispora sediminis||EU870859||98.6||SMP-S||Xestospongia sp. SYM12|
|FXJ6.014||GU002074||Nonomuraea turkmeniaca||NR_025111||98.2||M5||Xestospongia sp. SYM12|
|FXJ6.251||JF346446||Verrucosispora gifhornensis||AB546292||98.8||M5||Axinyssa sp. WNGB3|
|FXJ6.309||JN182146||Streptomyces sclerotialus||AB184071||98.2||M5-S||Hymeniacidon perleve MFDL|
|FXJ6.328||JF346459||Georgenia muralis||AB455495||96.8||M5||Hymeniacidon perleve MFDL|
|FXJ6.338||JF346462||Micromonospora pattaloongensis||AB275607||98.9||ISP 3||Reniochalina sp. SZDL|
|FXJ6.348||JF346468||Streptomyces laceyi||AB249944||98.9||DNBA||Reniochalina sp. SZDL|
|FXJ6.349||JF346469||Micromonospora pattaloongensis||AB275607||97.8||ISP 3||Reniochalina sp. SZDL|
|FXJ6.350||JF346470||Micromonospora pattaloongensis||AB275607||98.9||DNBA||Reniochalina sp. SZDL|
|Table 4. Numbers of positive isolates for antimicrobial activity and PKS I, PKS II and NRPS genes (percentage/tested isolates) from different sponges.|
*At least against one indicator microorganism.
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