J. Microbiol. Biotechnol. (2015), 25(1), 1–10 http://dx.doi.org/10.4014/jmb.1406.06041 Research Article Review jmb Sponge-Specific Unknown Bacterial Groups Detected in Marine Sponges Collected from Korea Through Barcoded Pyrosequencing S Jong-Bin Jeong1†, Kyoung-Ho Kim2†, and Jin-Sook Park1* 1Department of Biological Science and Biotechnology, Hannam University, Daejeon 305-811, Republic of Korea 2Department of Microbiology, Pukyong National University, Busan 608-737, Republic of Korea Received: June 13, 2014 Revised: September 29, 2014 The bacterial diversity of 10 marine sponges belonging to the species Cliona celata, an Accepted: October 13, 2014 unidentified Cliona species, Haliclona cinerea, Halichondria okadai, Hymeniacidon sinapium, Lissodendoryx isodictyalis, Penares incrustans, Spirastrella abata, and Spirastrella panis collected from Jeju Island and Chuja Island was investigated using amplicon pyrosequencing of the 16S First published online rRNA genes. The microbial diversity of these sponges has as of yet rarely or never been October 15, 2014 investigated. All sponges, except Cliona celata, Lissodendoryx isodictyalis, and Penares incrustans, *Corresponding author showed simple bacterial diversity, in which one or two bacterial OTUs occupied more than Phone: +82-42-629-8771; 50% of the pyrosequencing reads and their OTU rank abundance curves saturated quickly. Fax: +82-42-629-8769; Most of the predominant OTUs belonged to Alpha-, Beta-, or Gammaproteobacteria. Some of the E-mail: [email protected] OTUs from the sponges with low diversity were distantly (88%~89%) or moderately † These authors contributed (93%~97%) related to known sequences in the GenBank nucleotide database. Phylogenetic equally to this work. analysis showed that many of the representative sequences of the OTUs were related to the S upplementary data for this sequences originating from sponges and corals, and formed sponge-specific or -related clades. paper are available on-line only at The marine sponges investigated herein harbored unexplored bacterial diversity, and further http://jmb.or.kr. studies should be done to understand the microbes present in sponges. pISSN 1017-7825, eISSN 1738-8872 Copyright© 2015 by Keywords: Bacterial diversity, barcoded pyrosequencing, Chuja Island, Jeju Island, marine The Korean Society for Microbiology sponge and Biotechnology Introduction reveal so-called unidentified majorities [1] and unknown bacterial diversity in sponges [13, 36]. The massive parallel Sponges are well known as reservoirs of microbes; they 454 pyrosequencing, a next-generation sequencing method, are inhabited by high numbers of bacteria, which were has been applied to investigate the bacterial diversity in estimated to be up to 109 cells per milliliter of sponge tissue various environments [7, 30, 34] as well as in marine [14], accounting for as much as 50% of the biomass of the sponges [17, 18, 24, 33, 34, 45, 46]. sponges [31]. The microbes in sponges have various roles, Marine sponges harbor various kinds of bacteria, including as food sources [29], pathogenic agents [2], comprising a total of 25 bacterial phyla [34]. The most symbiotic residents [49], sponge skeleton stabilizers, diverse phyla contained in sponges are Proteobacteria, metabolic waste consumers, secondary metabolite producers Chloroflexi, and Poribacteria, but many less diverse phyla such [11], and nutrient cycle mediators [40]. as Acidobacteria, Actinobacteria, Cyanobacteria, Gemmatimonadetes, Numerous studies have been performed to reveal the Bacteroidetes, Spirochaetes, Firmicutes, Nitrospirae, TM7, diversity in sponges. Culture-dependent studies based on SBR1093, and OS-K have also been observed [34]. the isolation of pure cultures revealed various kinds of However, some sponges, known as low microbial bacteria, [44, 48] and novel strains were isolated and abundance (LMA) sponges, have low microbial content of proposed as new taxa [28, 51]. Many studies based on only about 105-106 cells per gram or milliliter [12]. LMA culture-independent methods have also been performed to sponges also have lower taxonomic diversity (less number January 2015 ⎪ Vol. 25⎪ No. 1 2Jeong et al. of taxa) than high microbial abundance (HMA) sponges, command of the Mothur package [32]. The QIIME package ver. and were reported to contain only up to five phyla in a 1.7.0 was used for to determine the OTUs and for their taxonomic study of five specimens of three different LMA sponges, assignment [22]. Sequence names were marked in accordance Callyspongia vaginalis, Niphates digitalis, and Raspailia topsenti with the samples and merged into one file. The uclust approach in [10]. the QIIME package was used for clustering the OTUs and the determination of representative sequences. A sequence similarity In the present study, the bacterial diversity in 10 sponges of 97% was used as the criterion for taxonomic assignments. The collected from Jeju Island and Chuja Island, Korea, was representative sequences of OTUs were compared with the investigated using pyrosequencing to reveal the unidentified 97_otus and the 97_otu_taxonomy files of the gg_13_05 version of bacterial diversity of sponges, including Cliona celata, the Greengenes database [27] and taxonomy was assigned using Haliclona cinerea, Lissodendoryx isodictyalis, Spirastrella abata, the RDP classifier [23]. Sequences related to chloroplasts and Spirastrella panis, and Penares incrustans, which have rarely mitochondria were discarded from further analyses. Diversity been explored to date. Most of the sponges, except Cliona estimators, including Chao1, Shannon, and Simpson indices, were celata, Lissodendoryx isodictyalis, and Penares incrustans, were determined for subsamples made with sequences of the same found to contain very simple diversity comparable to that numbers. Unifrac analysis [26] was performed to compare the of LMA sponges, and some of the predominant OTUs were samples. For the Unifrac analysis, similarities of 97%, 94%, 91%, found to be unrelated to known sequences. 88%, and 85% were used as criteria for OTU clustering. The PyNAST program [3] was used for the alignment of representative Materials and Methods sequences obtained from the clustering, after which the FastTree program [25] was used for construction of a phylogenetic tree using the aligned sequences. UPGMA trees were made from the Sample Collection and DNA Extraction Unifrac analysis. Some representative sequences were compared Sponge specimens were collected from Mueung-ri, Daejeong- against the nucleotide database of GenBank and related sequences eup, Seogwipo City, Jeju Province (Jeju Island), Korea (n =6) in were used for the construction of a phylogenetic tree. A neighbor- Feb. 2011 by scuba diving, and from around Chuja Island in the joining tree was constructed with the MEGA5 program [39], in South Sea of Korea (n = 4) in Nov. 2009 by scuba diving or from which the Kimura 2-parameter method [21] was used to calculate the intertidal region. Samples were collected aseptically and a distance matrix. delivered to the laboratory. Small pieces (about 1 cm3 in volume) of sponge tissues were washed with sterilized seawater, frozen at Results and Discussion -70oC for 24 h, and then lysophilized at -50oC, 0.033 Mbar for 24 h. The freeze-dried tissues were aseptically homogenized in a mortar. The G-spin genomic DNA extraction kit (Intron, South Sponges and Their Bacterial Communities Korea) was used for DNA extraction. All 10 specimens were so-called siliceous sponges (the class Demospongiae), which have spicules made out of silicon Barcoded Pyrosequencing dioxide [41]. Cliona celata is an ecologically/biotechnologically The V1 to V3 region of the 16S rRNA genes were amplified important species that is well known as an excavating and using primer sets (V1-9F: 5’-X-AC-GAGTTTGATCMTGGCTCAG-3’ cosmopolitan sponge [50]. The sterol composition and and V3-541R: 5’-X-AC-WTTACCGCGGCTGCTGG-3’; X indicates novel fatty acids were discovered from Haliclona cinerea the barcode sequences that were composed of various combinations sponges from the Black Sea [8, 19]. Lissodendoryx isodictyalis of six nucleotides to tag different samples). The Genome is an encrusting sponge with blue-gray color and a strong Sequencer FLX titanium (Roche, Germany) system was used for odor [35]. Lipids with cytotoxicity and other bioactivities pyrosequencing, according to the method in the manufacturer’s were isolated from Spirastrella abata sponges from Korea manuals (Macrogen, Korea). [15]. The diversity of cultured and uncultured bacteria of Bioinformatic Analysis of Reads Spirastrella abata and Spirastrella panis have been investigated After pyrosequencing, reads were sorted according to tags, and with restriction fragment length polymorphism and then the primer and tag sequences were cut out. Sequences less denaturing gradient gel electrophoresis (DGGE) [4, 16]. than 300 bp in size and with any ambiguous bases, “N”, were Bioactive triterpenoids and penaramides were discovered discarded from further analyses. Sequences with reverse from Penares incrustans sponges [37, 42]. However, the complement orientation were adjusted into the same orientation. latter five species have not yet been studied extensively. Sequences were aligned and the front and rear parts of sequences As far as we know, bacterial diversity (especially uncultured) that did not match with the other sequences were cut. Putative of sponges such as Cliona celata, Haliclona cinerea, Lissodendoryx chimera sequences were detected with the chimer.uchime