Culturable heterotrophic bacteria from the marine sponge Dendrilla nigra: isolation and phylogenetic diversity of actinobacteria
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Culturable heterotrophic bacterial composition of marine sponge Dendrilla nigra was analysed using different enrichments. Five media compositions including without enrichment (control), enriched with sponge extract, with growth regulator (antibiotics), with autoinducers, and complete enrichment containing sponge extract, antibiotics, and autoinducers were developed. DNA hybridization assay was performed to explore host specific bacteria and ecotypes of culturable sponge-associated bacteria. Enrichment with selective inducers (AHLs and sponge extract) and regulators (antibiotics) considerably enhanced the cultivation potential of sponge-associated bacteria. It was found that Marinobacter (MSI032), Micromonospora (MSI033), Streptomyces (MSI051), and Pseudomonas (MSI057) were sponge-associated obligate symbionts. The present findings envisaged that “Micromonospora–Saccharomonospora–Streptomyces” group was the major culturable actinobacteria in the marine sponge D. nigra. The DNA hybridization assay was a reliable method for the analysis of culturable bacterial community in marine sponges. Based on the culturable community structure, the sponge-associated bacteria can be grouped (ecotypes) as general symbionts, specific symbionts, habitat flora, and antagonists.
KeywordsSponge bacteria Culturable bacteria Bacterial endosymbionts Dendrilla nigra Actinobacteria
Most of the marine invertebrates harbor microorganisms that include bacteria, cyanobacteria, and fungi within their tissues where they reside in the extra- and intracellular space (Lafi et al. 2005; Vacelet and Donadey 1977; Wilkinson 1978; Wilkinson et al. 1981). The presence of large amounts of microorganisms within the mesohyl of many demosponges has been well documented (Hentschel et al. 2002; Imhoff and Stöhr 2003). Sponge mesohyl was referred as “micro-environments” providing a broad variety of ecological niches (Thiel et al. 2007). Bacteria can contribute up to 40% of the sponge biomass (equal to about 108 to 109 bacteria g tissue−1) and are probably permanently associated with the host sponge unless they are disturbed by external stress factors (Friedrich et al. 2001; Thoms et al. 2003; Webster and Hill 2001). Sponges are filter feeders and consume microorganisms from the inhaled seawater by phagocytosis. The relationships of marine invertebrates and microorganisms that may serve as food or that lives either permanently or temporarily inside of marine macroorganisms are highly complex and far from being understood (Steinert et al. 2000). The sponge–symbiont relationship could be categorized as obligatory mutualism (i.e., the symbionts play an essential role in the metabolism of their host), facultatively mutualism (they have a beneficial effect on their host, but the host will survive without the symbiont), or commensalism (they are present without providing obvious beneficial effects to their host). In all cases, it is assumed that the sponge host provides a sheltered habitat for their symbionts. A further distinction is made between epibionts (microorganisms living on the sponge surface) and endosymbionts (microorganisms that either live in the sponge mesohyl or inside the sponge cells) (Osinga et al. 1998). Microorganisms not only serve as food for filter feeders but may, perhaps, also be involved in the biosynthesis of secondary metabolites that are used for the discovery of novel drugs (Abrell 1997; Debitus et al. 1998; Perovic et al. 1998). In this background, the present study was initiated to explore the culturable heterotrophic bacterial composition of marine sponge Dendrilla nigra, which showed broad spectrum of bioactivity (Selvin and Lipton 2004a, b). In this study, we report phylogenetic analysis of cultured actinobacteria and DNA hybridization assay to target sponge-specific bacteria among the most common groups.
Materials and methods
Collection of D. nigra
The host sponge D. nigra was collected from southwest coast of India by SCUBA diving at 12 m depth. The collection site, Vizhinjam coast, is located at 8021′N and 7700′E on the west coast of India comprising of sandy beaches and irregularly distributed intertidal rocky substratum. The temperature of seawater was 25°C. Morphological observation of collected specimens indicated that a unique macrosymbiont, Indian brittle star Amphiura carchara, was invariably associated with all the specimens. Therefore, brittle star was separated from the host sponge, enclosed in separate sterile bags, and then transferred to the laboratory on ice. Only unbroken samples were used for microbiological analysis to avoid cross contamination. The specimens kept for 2 h in sterilized aged seawater to remove loosely associated microorganisms from inner and outer sponge surfaces. It has been hypothesized that this process may eliminate non-associated bacteria from the host sponge by digestion. Environmental water representing the sponge habitat was taken prior to sponge sampling and filled up in 1 l sterilized glass bottles. The preserved (frozen) specimens, photographs, and videograph of the collection process and habitat were retained in the laboratory for future reference. Specimens were identified and/or confirmed with the help of renowned sponge taxonomist P.A. Thomas, Scientist Emeritus, Central Marine Fisheries Research Institute, India.
Isolation and culture of sponge-associated bacteria
One cubic centimeter of sponge tissue was excised from the middle of the whole sponge using a sterile scissors. The excised portion was washed three times with sterile seawater to remove any bacteria within current canals and then the tissue was homogenated using a tissue homogenizer (Omni). The resultant homogenate was serially diluted with sterilized aged seawater and preincubated at 40°C for 1 h for the activation of dormant and inactive cells. The resultant aliquot was plated on various isolation media and incubated at 25°C in dark aerobic conditions except the anaerobic agar plates until visible colonies appeared. In addition to marine sponge agar (MSA) (Selvin et al. 2004), standard media such as modified marine agar (MMA), seawater agar (SA), modified nutrient agar (MNA), halophilic agar (HA), actinomycetes agar (AA), starch casein agar (CSA), raffinose-histidine medium (RH), fluid-thioglycollate medium (FT), malt agar (MA), Emerson Agar (EA), TCBS Agar (TA), Pseudomonas agar (PA), and anaerobic agar (ANA). The media composition was rationalized and used for the isolation of sponge-associated bacteria. The medium composition per liter of MSA was raffinose 1.0 g, l-histidine 1.0 g, FeSO4 · 7H2O 0.01 g, K2HPO4 1.0 g, CaCO3 0.02 g, MgSO4 · 7H2O 0.5 g, agar 15 g, and NaCl 20 g. The medium was enriched with 10% each of aqueous and organic host sponge extracts, respectively. The ingredients per liter included in the modified media are given below:
Peptic digest of animal tissue 0.5 g, yeast extract 0.1 g, ferric citrate 0.1 g, sodium chloride 20 g, magnesium chloride 0.8 g, sodium sulfate 0.32 g, calcium chloride 0.18 g, potassium chloride 0.5 g, sodium bicarbonate 0.2 g, potassium bromide 0.08 g, strontium chloride 0.03 g, boric acid 0.02 g, sodium silicate 0.004 g, sodium fluorate 0.002 g, ammonium nitrate 0.002 g, disodium phosphate 0.008 g, sponge organic extract—1%, and dextrose 0.05 g.
Peptic digest of animal tissue 0.5 g, yeast extract 0.15 g, beef extract 0.15 g, magnesium chloride 0.88 g, dextrose—0.05 g and sodium chloride 20 g.
The composition as in MNA except sodium chloride and the composition was prepared in 1 l aged seawater instead of double distilled water.
NaCl 250.0 g, MgSO4 · 7H2O 2.5 g, casamino acids 1.0 g, yeast extract 1.0 g, protease peptone 0.5 g, trisodium citrate 0.3 g, and KCl 2.0 g.
Beef heart infusion solids 1.0 g, tryptose 1.0 g, casein enzymic hydrolysate 0.4 g, yeast extract 0.5 g, dextrose 0.5 g, l-cysteine hydrochloride 0.1 g, starch 0.1 g, sodium chloride 20.0 g, monopotassium phosphate 1.5 g, ammonium sulfate 1.0 g, magnesium sulfate 0.2 g, and calcium chloride 1.0 g.
Casein 1.0 g, starch 1.0 g, and seawater 37.0 g.
Raffinose 1.0 g, l-histidine 1.0 g, MgSO4 0.5, FeSO4 0.01 g, and NaCl 20 g.
Casein enzymic hydrolysate 1.5 g, yeast extract 0.5 g, dextrose 0.5 g, sodium chloride 20 g, l-cystine 0.5 g, sodium thioglycollate 0.5 g, and resazurin sodium 0.001 g.
Malt extract 2.0 g, dextrose 0.5 g, and NaCl 20 g.
Peptic digest of animal tissue 0.4 g, beef extract 0.4 g, yeast extract 1.0 g, dextrose 0.1 g, and sodium chloride 20 g.
Peptone 1.5 g, yeast extract 0.6 g, sodium thiosulphate 1.0 g, sodium citrate 1.0 g, synthetic detergent II 0.2 g, sucrose 2.0 g, sodium chloride 20 g, ferric citrate 1.0 g, bromo thymol blue 0.04 g, and thymol blue 0.04 g.
Casein enzymic hydrolysate 1.0 g, proteose peptone 1.0 g, dipotassium phosphate 0.15 g, magnesium sulfate 0.15 g, and sodium chloride 20 g.
Casein enzymic hydrolysate 1.2 g, peptic digest of animal tissue 0.5 g, yeast extract 0.3 g, beef extract 0.3 g, corn starch 0.1 g, dextrose 0.1 g, sodium chloride 20.0 g, dithiothreitol 0.1 g, l-cystine hydrochloride 0.5 g, vitamin K1 0.01 g, sponge organic extract 1% and dextrose 0.05 g. The media prepared in 1-l double distilled water and agar 20 g added to obtain solid media.
Another batch of these media were enriched with selective inducers/regulator containing aqueous host sponge extract 2% (sponge agar exempted), organic extract 1%, cAMP (Sigma) 1 μM, alpha-butyrolactone 0.1 μM, nalidixic acid 10 μM, NaCl-2%, and Nystatin 10 μM (pH 7.8). The media ingredients were sterilized separately at 121°C, 15 Ibs for 10 min, and the selective enrichments were filtered through 0.2 μ filter (Millipore®) and added to the sterilized ingredients. The host sponge extract was used as “enhancers” of sponge-specific bacterial cultivation potential, the antibiotics, nadixic acid, and nystatin screened selectively against the surrounding seawater bacteria and fungi, respectively, was used as “regulator” of fast growing bacteria and alpha-butyrolactone was used as “autoinducer” which increase the cultivation potential of unculturable bacteria. Using different enrichments, five media compositions were obtained including: (1) media without enrichment (control), (2) enriched with sponge extract, (3) enriched with nystatin, (4) enriched with alpha-butyrolactone, and (5) complete enrichment containing sponge extract, nystatin, and alpha-butyrolactone. One gram tissue of brittle star was processed in the same way as described for the host sponge. Habitat water samples were serially diluted in sterile-aged seawater and plated for the analysis of habitat microflora. Growth of associated bacteria and free living bacteria in the habitat water was determined in terms of colony forming units (CFU). Only consistent morphotypes in subsequent subcultures were considered, and representatives of the most abundant morphotypes were chosen for DNA hybridization assay. Morphological biochemical and physiological characteristics of bacterial isolates were determined as per standard protocols (Cappuccino and Sherman, 2004; MacFaddin 1981). The test characteristics were selected on the basis of appropriate previous step of the identification scheme. The antagonistic activity was determined by cross-streaking followed by Burkholder agar diffusion assay (Burkholder et al. 1966).
Isolation of DNA and sequencing
Genomic DNA of bacterial symbionts and freeze-dried tissue of host sponge were extracted by the methods as described in earlier reports (Enticknap et al. 2006; Lopez et al. 1999; Pitcher et al. 1989). Both universal and genus-specific primers were used for the amplification of DNA. The reaction mixture was prepared in a total volume of 50 μl including 100 ng of template DNA, 5 μl of 10 × PCR reaction buffer (100 mM Tris–HCl, pH 9.0, 50 mM KCl, 1.5 mM MgCl2, 0.1% (w/v) Triton X-100, 0.2 ml BSA, 2.5 U of Taq DNA polymerase (Finnzyme), 200 μM of each dNTP, 2 mM forward primer, and 2 mM reverse primer. The complete reaction mixture was incubated in a gradient thermocycler (Eppendorf). The PCR temperature profile used was: 94°C for 3 min, then 30 cycles consisting of 94°C for 1 min, 52°C for 2.0 min, 72°C for 2 min, and finally an extension step at 72°C for 6 min. PCR products were analyzed by electrophoresis on 0.8% (w/v) agarose TAE-gels. PCR products were purified using a PCR purification kit (Genei), then cloned into pGEMT vector (Promega) following the manufacturer’s instructions. The ligated product was transformed into Escherichia coli competent cells, and the transformed cells were obtained on ampicillin, X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranose), and IPTG (isopropyl-β-d-thiogalactoside) containing LB agar plates. Positive clones were picked by blue/white selection and checked for size of the right insert by PCR.
The 16S ribosomal RNA gene sequence obtained from the actinobacterial isolates was compared with other representative sequences by using NCBI megaBLAST. Taxonomic affiliation of the sequences was retrieved from classifier program of ribosomal database project (RDP-II) (Wang et al. 2007). RDP-II hierarchy was based on the new phylogenetically consistent higher-order bacterial taxonomy proposed by Garrity et al. (2007). Multiple alignments of these sequences were carried out by ClustalW 1.83 version of EBI (www.ebi.ac.uk/cgi-bin/clustalw/) with 0.5 transition weight. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) is shown next to the branches (Felsenstein, 1985). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method (Tamura et al. 2004) and are in the units of the number of base substitutions per site. All positions containing alignment gaps and missing data were eliminated only in pairwise sequence comparisons (Pairwise deletion option). Phylogenetic trees were constructed in MEGA 4 version (www.megasoftware.net) using neighbor-joining (NJ), minimum evolution (ME), and unweighted pair group method with arithmetic mean (UPGMA) algorithms (Tamura et al. 2007). Interior NJ and ME trees were constructed by using kimura 2-parameter model. The sequence data of the representative strains of each group were deposited in GenBank under the accession numbers EF417875, EF428028, EF428029, EF428030, EF428031, EF428032, EF428033, EU19923, EU199242, EU199243, EU417875, EU563352, and EU563351.
DNA hybridization assay
Oligonucleotide probes used in this study
Sequences 5′ → 3′
Bacterial 16S rRNA (338–335)
GCT GCC TCC CGT AGG AGT
ACT CCT ACG GGA GGC AGC
MSI026 16S rRNA (734–754)
MSI032 16S rRNA (82–102)
MSI033 16S rRNA (703–723)
MSI039 16S rRNA (605–625)
MSI042 16S rRNA (46–66)
MSI051 16S rRNA (792–812)
MSI057 16S rRNA (171–191)
Results and discussion
Total culturables (TC) and group specific colony counts
HS (cfu/cm2 area)
BS (cfu/g tissue)
1.45 × 105
9.6 × 104
1.12 × 105
2.2 × 104
3.9 × 103
2.4 × 104
6.3 × 104
2.6 × 104
1.7 × 104
2.8 × 103
2.4 × 104
2.9 × 103
1.6 × 104
1.4 × 104
4.8 × 104
It has been suggested that the growth media should resemble as much as possible the conditions that prevail inside the sponge mesohyl. Recently, it has been realized the production of acyl homoserine lactones (AHLs) by bacteria associated with marine sponges (Michael et al. 2004). Many gram-negative bacteria utilize AHL-mediated signaling systems to communicate with one another (Fuqua et al. 2001). These systems involve the production of low-molecular weight molecules that accumulate with increasing bacterial numbers and thus provide an index of population density. When a threshold bacterial density (and corresponding AHL concentration) is reached, AHLs interact with transcriptional activators to trigger the expression of target genes. Many terrestrial bacteria produce AHLs, yet, beyond the well-characterized Vibrio fischeri-squid symbioses (Boettcher and Ruby 1995), relatively little is known about the occurrence of AHLs in marine bacterial endosymbionts. However, it has been realized that the addition of AHLs and cyclic AMP (Bruns et al. 2002) or siderophores (Guan et al. 2000) to marine growth media increased bacterial culturability and growth, respectively. The present findings envisaged that the cultivation potential of sponge bacteria could be increased considerably by the addition of selective inducers (AHLs and sponge extract) and regulators (antibiotics). In a previous report, 35% of the supplemented media demonstrated CFU recoveries from marine sponges that were greater than those of the unamended control plates (Olson et al. 2000). New fermentation technologies were developed for symbionts that are not cultivable using the existing media (Piel et al. 2005). Some sponge isolates have been cultured (Osinga et al. 1998), but it remains uncertain whether these are associated bacteria or symbionts. Therefore, in the present study, obligate symbionts were detected using DNA hybridization assay. This is the first report that envisages the culturable obligate symbionts of a marine sponge from Indian coast.
Blast search and phylogenetic analysis based on 16S rRNA gene sequences
Detection and grouping of sponge-associated bacteria
Detection of probe specific bacteria in HS, BS, and HW samples
Value ratio sample/blanka
EUB338b Positive control
NON338c Negative control
Association of culturable actinobacteria including Micromonospora and Streptomyces was reported from Haliclona sp. (Jiang et al. 2007). Micromonospora have been isolated previously from a marine sponge Hymeniacidon perleve by Zhang et al. (2006). However, the association of Sccharomonospora was seldom reported from marine sponges. Maldonado et al. (2005) described the “Micromonospora–Rhodococcus–Streptomyces” group seems to be ubiquitous in cultured actinobacteria from marine environments. Based on the present findings, “Micromonospora–Saccharomonospora–Streptomyces” group seems to be major culturable actinobacteria in marine sponge D. nigra. Domain-specific universal bacterial primers were used to amplify the 16S rDNA gene from genomic DNA that had been extracted from sponges and the surrounding water of Caribbean sponge, Chondrilla nucula (Hill et al. 2006). It was also reported that highly diverse bacterial communities including some percentage appear to be specialized for the sponge habitat. Burja et al. (1999) reported a highly diverse assemblage of more than 200 heterotrophic bacteria isolated from the Great Barrier Reef sponge Candidaspongia flabellate based on biochemical characterization and this finding was confirmed by the 16S rRNA sequence analysis. In the present study, the DNA hybridization assay was found to be a reliable alternate over other hybridization methods.
The present study was aimed to retrieve sponge-associated and sponge-specific bacteria by novel cultivation approaches. The media rationalized with host sponge extract and alpha-butyrolactone as autoinducer increase the cultivation potential of sponge-associated bacteria. The supplementation of host sponge extract with antibiotics selectively screened against the surrounding seawater (habitat) bacteria increase the scope of retrieving sponge-specific bacteria in culture. Albeit the present study demonstrated the possible retrieval of sponge-associated and sponge-specific bacteria in culture, the number of isolates retrieved might perhaps be a small percent of total microbial symbionts. It has been established that cultivation approaches could retrieve 1% of total microbial symbionts and the remaining uncuturable majority require culture independent approaches. The metagenomic approaches to explore the unculturable microbial majority of D. nigra is under investigation in our laboratory. A limitation of the molecular approach to study sponge-associated bacteria is the possibility of contamination by bacteria from the surrounding seawater and/or associated with invertebrates living inside the sponge (Wichels et al. 2006; Thiel et al. 2007). The present findings demonstrated the possibility of targeting the cultured sponge-specific bacteria by DNA hybridization assay. Meyer and Kuever (2007) demonstrated that the microbial consortium of marine sponge Polymastia cf. corticata and the ambient bacterioplankton were distinctly different, indicative for the low impact of the surrounding seawater on the sponge-microbe associations. However, in contrast, the present findings evidenced that the sponge-associated bacteria represents transient bacterial populations in the surrounding seawater.
Based on the culturable community structure, the sponge-associated bacteria can be typed as general symbionts, specific symbionts, habitat flora, and antagonists. Based on the local distribution of phylotypes of endosymbionts in the host sponge, the microbial symbionts were classified as “sponge associate,” “specialist,” and “generalist” (Meyer and Kuever 2007). Sponges are filter-feeders, sequestrate seawater-derived microorganisms that might be enriched by the stable and nutritionally rich microhabitat and become part of the sponge-associated microbiota. The host sponge might provide distinct microenvironments as ecological niches for the different bacterial and archaeal populations. The supplementation of host sponge extract might create such nutritional niche required for the growth of sponge-specific bacteria. The present findings envisaged that majority of culturable bacteria are actinobacteria. In conclusion, the cultivation efficiency of sponge-associated bacteria could be increased considerably using autoinducers and regulators as selective enrichment. The present study is the first report on the cultivation and ecotyping of bacteria associated with D. nigra. The DNA hybridization assay appeared as unique assay for the analysis of culturable bacterial community in marine sponges.
JS is thankful to the International Foundation for Science (IFS), IFS project RGA No. F/3776-1, Sweden, and Ministry of Earth Sciences (MoES), OASTC (Marine Microbiology) for funding. JS also thank Director, IWST (Indian Council for Forestry Research and Education), Bangalore, for facilities and support during initial phase of the project. RG is thankful to the Council of Scientific and Industrial Research (CSIR), New Delhi, for the award of Senior Research Fellowship.
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