OPEN The ISME Journal (2017) 11, 2465–2478 www.nature.com/ismej ORIGINAL ARTICLE Metagenomic binning of a marine sponge microbiome reveals unity in defense but metabolic specialization Beate M Slaby1,2, Thomas Hackl3, Hannes Horn1,2, Kristina Bayer1 and Ute Hentschel1,4 1RD3 Marine Microbiology, GEOMAR Helmholtz Centre for Ocean Research Kiel, Kiel, Germany; 2Department of Botany II, Julius-von-Sachs Institute for Biological Science, University of Würzburg, Würzburg, Germany; 3Department of Civil and Environmental Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA and 4Christian-Albrechts University of Kiel, Kiel, Germany Marine sponges are ancient metazoans that are populated by distinct and highly diverse microbial communities. In order to obtain deeper insights into the functional gene repertoire of the Mediterranean sponge Aplysina aerophoba, we combined Illumina short-read and PacBio long-read sequencing followed by un-targeted metagenomic binning. We identified a total of 37 high-quality bins representing 11 bacterial phyla and two candidate phyla. Statistical comparison of symbiont genomes with selected reference genomes revealed a significant enrichment of genes related to bacterial defense (restriction-modification systems, toxin-antitoxin systems) as well as genes involved in host colonization and extracellular matrix utilization in sponge symbionts. A within-symbionts genome comparison revealed a nutritional specialization of at least two symbiont guilds, where one appears to metabolize carnitine and the other sulfated polysaccharides, both of which are abundant molecules in the sponge extracellular matrix. A third guild of symbionts may be viewed as nutritional generalists that perform largely the same metabolic pathways but lack such extraordinary numbers of the relevant genes. This study characterizes the genomic repertoire of sponge symbionts at an unprecedented resolution and it provides greater insights into the molecular mechanisms underlying microbial-sponge symbiosis. The ISME Journal (2017) 11, 2465–2478; doi:10.1038/ismej.2017.101; published online 11 July 2017 Introduction Webster and Thomas, 2016). 16S rRNA gene ampli- con studies discovered an unusually high phylum- Marine sponges (Porifera) are evolutionary ancient level diversity and stability of microbial associations metazoans dating back to Precambrian times in marine sponges comprising phototrophic as well (Li et al., 1998; Love et al., 2009). By filtering as heterotrophic symbionts (Schmitt et al., 2012a; extensive volumes of seawater—up to thousands of — Easson and Thacker, 2014; Thomas et al., 2016; liters per kg sponge daily (Reiswig, 1974) they take Webster and Thomas, 2016). The sponge microbiome in food bacteria, but also potential pathogens, toxins includes as many as 52 microbial phyla and and physical stress factors (De Goeij et al., 2009). candidate phyla with the diversity and abundance Rapid cell turnover rates accompanied by extensive varying between sponge species (Webster and detritus production are likely a means of avoiding Thomas, 2016). The most dominant symbiont groups permanent, stress-induced damage to the sponge (De belong to the phyla Proteobacteria (mainly gamma- Goeij et al., 2009; Alexander et al., 2014). In line with and alphaproteobacteria), Actinobacteria, Chloro- the holobiont concept (Bordenstein and Theis, 2015), flexi, Nitrospirae, Cyanobacteria, candidatus phylum the highly diverse and distinct symbiotic microbial Poribacteria, and Thaumarchaea (Webster and communities of marine sponges are thought to Thomas, 2016). play a crucial role in their evolutionary success Comparisons of sponge-associated and seawater (Easson and Thacker, 2014; Tian et al., 2014; microbial consortia have identified a number of genomic features that seem to facilitate bacterial Correspondence: U Hentschel, RD3 Marine Microbiology, GEO- adaptation to a symbiotic existence within sponges, MAR Helmholtz Centre for Ocean Research Kiel, Düsternbrooker for example, transposable elements, defense Weg 20, Kiel, Germany. mechanisms, and eukaryote-like proteins (Thomas E-mail: [email protected] Received 24 December 2016; revised 7 May 2017; accepted 21 May et al., 2010; Fan et al., 2012; Hentschel et al., 2012; 2017; published online 11 July 2017 Horn et al., 2016). Studies on individual clades of Marine sponge symbionts: united in defense but specialized in metabolism BM Slaby et al 2466 the microbial consortium have revealed specific Materials and methods features of sponge symbionts, such as adaptations of the lipopolysaccharide by the cyanobacterium Sample collection ‘Candidatus Synechococcus spongiarum’ presum- Aplysina aerophoba specimens were collected from ably to avoid host phagocytosis (Gao et al., 2014; the Mediterranean Sea near Piran, Slovenia Burgsdorf et al., 2015) and specific patterns for (45.517680, 13.567991). One specimen was collected carbon degradation by Poribacteria (Kamke et al., in May 2013 for Illumina sequencing and one 2013). specimen was collected in May 2014 for PacBio PacBio long-read sequencing is widely used in sequencing. Both were collected from ca. 5 m depth isolate genomics as a stand-alone tool or in and transported to the laboratory in natural seawater combination with Illumina short-read sequencing at ambient temperature. Sponge pinacoderm (outer (for example, Beims et al., 2015; Koren and layer) and mesohyl (inner core), visually distinguish- Phillippy, 2015; Ricker et al., 2016). The error- able by the reddish-greenish color of the prone PacBio reads need to be corrected either with cyanobacteria-containing pinacoderm, were sepa- themselves—if sufficient sequencing depth is pro- rated with a sterile scalpel blade and microbial cell vided—or with Illumina reads of far lower error-rate enrichment was performed by differential centrifu- (Koren et al., 2012; Ono et al., 2013). The combina- gation (Fieseler et al., 2006). These sponge- associated prokaryotes (SAPs) were frozen with tion of PacBio and Illumina sequencing data in − hybrid assemblies enables the closure of assembly 15% glycerin at 80 °C. gaps, for example, by spanning over long repeats, to merge contigs and thereby to reconstruct the genome architecture (Koren et al., 2012). In meta- DNA extraction and sequencing genomics, the Illumina-PacBio hybrid assembly DNA of sponge-associated prokaryotes (SAPs) approach has recently been shown to improve the obtained from either pinacoderm or mesohyl tissue quality of assemblies (Frank et al.,2016;Tsaiet al., (three technical replicates each) was extracted with 2016). Although the improvements in targeted the FastDNA SPIN Kit for Soil (MP Biomedicals, binning of the dominant members of the micro- Santa Ana, CA, USA). Different cell lysis protocols biomes in these studies have been demonstrated were applied for each triplicate to obtain differential (Frank et al., 2016; Tsai et al., 2016), un-targeted sequencing coverage for downstream binning as binning and performance for less abundant mem- previously described (Albertsen et al., 2013; bers of the microbial communities have not been Alneberg et al., 2014): (i) bead beating, following evaluated. the manufacturer’s protocol, (ii) freeze-thaw cycling Even though considerable metagenomic informa- (3 cycles of 20 min at − 80 °C and 20 min at 42 °C), (iii) proteinase K digestion for 1 h at 37 °C (TE buffer tion from sponge microbiomes has been accrued, − only few symbiont genomes have been recon- with 0.5% SDS and proteinase K at 100 ng ml 1 final structed by single-cell genomics or binning (Siegl concentration). Metagenomic DNA was sequenced et al., 2011; Kamke et al., 2013; Gao et al., 2014, on an Illumina HiSeq2000 platform (150-bp paired- Burgsdorf et al., 2015). Therefore, correlations end reads) and quality filtered at the DOE Joint between phylogeny and function have only rarely Genome Institute (Walnut Creek, CA, USA) follow- been possible. In the present study, we aimed at ing the JGI sequencing and the data processing obtaining a larger number and greater diversity of pipeline (Markowitz et al., 2012). Additionally, V4 sponge symbiont genomes. We present the first iTag sequences were obtained by Illumina MiSeq metagenomic hybrid assembly derived from Illu- sequencing and analyzed in the respective iTagger mina short-read and the PacBio long-read data with pipeline at JGI (for more information, see https:// subsequent un-targeted differential coverage bin- bitbucket.org/berkeleylab/jgi_itagger and http://jgi. ning. The highly complex microbiome of the doe.gov/wp-content/uploads/2013/05/iTagger-meth Mediterranean sponge Aplysina aerophoba was ods.pdf). For the PacBio data set, DNA was extracted used towards this goal. By applying an un-targeted with the above-mentioned kit following the manu- binning technique, we aimed to include also the facturer’s protocol (cell lysis by bead beating) and less abundant members of the microbial commu- sequenced on a PacBio RS II platform using 8 nity. We provide statistical evidence for gene SMRT cells by GATC Biotech (Konstanz, Germany). networks that are enriched in the symbiont gen- omes over selected reference genomes and we discuss the role of these genomic adaptations in Assembly, binning, and annotation context of a symbiotic existence in the sponge Illumina reads were coverage-normalized with matrix. Furthermore, a comparison between sym- bbnorm of BBMap v. 34 (https://sourceforge.net/ biont genomes revealed a specialization into three projects/bbmap/) at default settings. PacBio