Single-Cell Genomics Reveals Complex Carbohydrate Degradation Patterns in Poribacterial Symbionts of Marine Sponges
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The ISME Journal (2013) 7, 2287–2300 & 2013 International Society for Microbial Ecology All rights reserved 1751-7362/13 www.nature.com/ismej ORIGINAL ARTICLE Single-cell genomics reveals complex carbohydrate degradation patterns in poribacterial symbionts of marine sponges Janine Kamke1, Alexander Sczyrba2,3, Natalia Ivanova2, Patrick Schwientek2, Christian Rinke2, Kostas Mavromatis2, Tanja Woyke2 and Ute Hentschel1 1Department of Botany II, Julius-von-Sachs Institute for Biological Sciences, University of Wuerzburg, Wuerzburg, Germany; 2Department of Energy Joint Genome Institute, Walnut Creek, CA, USA and 3Center for Biotechnology, Bielefeld University, Bielefeld, Germany Many marine sponges are hosts to dense and phylogenetically diverse microbial communities that are located in the extracellular matrix of the animal. The candidate phylum Poribacteria is a predominant member of the sponge microbiome and its representatives are nearly exclusively found in sponges. Here we used single-cell genomics to obtain comprehensive insights into the metabolic potential of individual poribacterial cells representing three distinct phylogenetic groups within Poribacteria. Genome sizes were up to 5.4 Mbp and genome coverage was as high as 98.5%. Common features of the poribacterial genomes indicated that heterotrophy is likely to be of importance for this bacterial candidate phylum. Carbohydrate-active enzyme database screening and further detailed analysis of carbohydrate metabolism suggested the ability to degrade diverse carbohydrate sources likely originating from seawater and from the host itself. The presence of uronic acid degradation pathways as well as several specific sulfatases provides strong support that Poribacteria degrade glycosaminoglycan chains of proteoglycans, which are important components of the sponge host matrix. Dominant glycoside hydrolase families further suggest degradation of other glycoproteins in the host matrix. We therefore propose that Poribacteria are well adapted to an existence in the sponge extracellular matrix. Poribacteria may be viewed as efficient scavengers and recyclers of a particular suite of carbon compounds that are unique to sponges as microbial ecosystems. The ISME Journal (2013) 7, 2287–2300; doi:10.1038/ismej.2013.111; published online 11 July 2013 Subject Category: Microbe-microbe and microbe-host interactions Keywords: marine sponge; symbiont; carbohydrate degradation; extracellular matrix; single-cell genomics Introduction ‘mesohyl’, and can make up 35% of the sponge body mass (Vacelet, 1975). Collectively, representatives of Marine sponges (phylum Porifera) are the most more than 30 bacterial phyla and both archaeal ancient extant metazoans with fossil records, indi- lineages have so far been found in sponges from cating their emergence more than 600 million years various geographic locations (Webster et al., 2010; ago (Love et al., 2009). These animals are sessile Schmitt et al., 2012; Simister et al., 2012). The filter feeders with an enormous filtering capacity microbial diversity of marine sponges is well inves- that is known to affect nutrient concentrations in the tigated (Taylor et al., 2007), and the collective surrounding environment (Gili and Coma, 1998; repertoire of ‘omics’ approaches has been instrumen- Maldonado et al., 2005, 2012). In addition to their tal to shed light on the functional genomic traits of the evolutionary and ecological significance, sponges collective sponge microbiome (Thomas et al., 2010; have attracted recent scientific attention owing to Fan et al., 2012; Liu et al., 2012; Radax et al., 2012). their specific and unique microbiology (Hentschel However, community-wide approaches do not pro- et al., 2012). The microbial biomass in sponges is vide sufficient information about functions of specific located in the extracellular matrix, the so-called symbiont clades. Providing a thorough understanding of symbiont function is further complicated by the Correspondence: U Hentschel, Department of Botany II, Julius- fact that many sponge symbiont lineages remain von-Sachs Institute for Biological Sciences, University of uncultivated, such as for the many candidate phyla Wuerzburg, Julius-von-Sachs-Platz 3, Wuerzburg 97082, Germany. E-mail: [email protected] found in these animals (Schmitt et al., 2012). Received 26 February 2013; revised 4 June 2013; accepted 4 June One such candidate phylum, termed Candidate 2013; published online 11 July 2013 phylum Poribacteria, was originally discovered and Carbohydrate degradation in Poribacteria J Kamke et al 2288 described in marine sponges (Fieseler et al., 2004). Dickinson, Heidelberg, Germany) as described pre- Poribacteria are widely distributed and highly viously (Siegl and Hentschel, 2010). For cell lysis abundant in sponge species around the world and whole genome amplification (WGA) by multiple (Fieseler et al., 2004; Lafi et al., 2009; Schmitt displacement amplification, we followed the same et al., 2011, 2012), and also occur freely in seawater, procedure as Siegl et al. (2011). To identify phylo- albeit at very low abundances (Pham et al., 2008; genetically WGA products and check for possible Webster et al., 2010; Taylor et al., 2013). They were contamination, we screened the WGA products shown to be affiliated with the Planctomycetes, obtained by polymerase chain reaction (PCR) using Verrucomicrobia, Chlamydiae (PVC) superphylum 16S or 18S rRNA gene primers targeting Eubacteria, (Wagner and Horn, 2006). Poribacteria are vertically Archaea, Poribacteria and Eukaryotes, as described transmitted via reproductive stages (Schmitt et al., previously (Siegl and Hentschel, 2010). Poribacteria- 2008, Webster et al., 2010). Owing to their abun- positive WGA products were additionally screened dance and diversity within sponges, Poribacteria with the degenerated PCR primer pair 27f-B and can be regarded as a model sponge symbiont. 1492r-B (Cho and Giovannoni, 2004) that covers Single-cell genomics has become the most useful Eubacteria more broadly and enables to obtain tool to investigate the genomic repertoire of distinct longer 16S rRNA gene sequences from Poribacteria. uncultivated microbial symbionts (Kamke et al., Subsequent cloning of PCR products, restriction 2012) and other microorganisms (Woyke et al., 2009; fragment length polymorphism analysis and Sanger Yoon et al., 2011; Stepanauskas, 2012). It also has sequencing was conducted to confirm the presence previously been successfully applied to a poribac- of a single cell using the same procedures as Siegl terial cell (Siegl et al., 2011). Unlike other ‘omics’ and Hentschel (2010). approaches, this method can connect phylogenetic WGA products that originated from single identity with the functional potential of unculti- poribacterial cells were then subjected to another vated microbial organisms, even from high diversity round of multiple displacement amplification under environments. Here we used single-cell genomics to the same conditions as stated above. Before genome analyze five poribacterial cells from the Mediterra- sequencing, we conducted a S1 nuclease treatment nean sponge Aplysina aerophoba, expanding the and DNA purification as described by Siegl et al. existing data set from one (Siegl et al., 2011) to a (2011). Five SAGs were selected in the PCR screening total of six poribacterial single-amplified genomes process and subjected to whole genome sequencing: (SAGs). We provide an in-depth genomic analysis of Candidatus Poribacteria WGA-3G, WGA-4C, WGA- one of the main symbiont lineages in the complex 4CII, WGA-4E and WGA-4G (hereafter referred as 3G, microbiota of marine sponges. The property of 4C, 4CII, 4E and 4G, respectively). These were carbohydrate degradation emerged as the most complemented by one poribacterial SAG sequence common feature among the analyzed genomes. We from an earlier study by Siegl et al. (2011), Candidatus therefore focused on carbohydrate degradation Poribacteria WGA-A3 (hereafter referred as 3A). The potential of Poribacteria in this study and discussed existing assembly for this SAG was used for annota- the results in context of a nutritional basis of the tion and further analyses as described below. sponge–microbe symbiosis. Genome sequencing, assembly and annotation Materials and methods A detailed description of all steps of genome Sample collection and processing sequencing, assembly, annotation and quality Samples of the marine sponge A. aerophoba were checks can be found in Supplementary Text S1. collected in September 2009 by scuba diving to a Briefly, a combination of Illumina and 454 pyrose- depth of 5–12 m at the Coast of Rovinj, Croatia quencing was conducted for double displacement (451080N, 131640E). The animals were transported to amplification products of SAGs 4C, 4E and 4G at the University of Wuerzburg (Wuerzburg, Germany) LGC Genomics (Berlin, Germany) and the DOE Joint and kept in seawater aquaria until further processing Genome Institute (JGI, Walnut Creek, CA, USA). within 1 week of collection. Fresh sponge samples SAGs 3G and 4CII were also sequenced at JGI using were used for extraction of sponge-associated pro- Illumina HiSeq2000 technology only. Illumina karyotes using an established protocol of tissue sequences were normalized using DUK, a filtering disruption, density centrifugation and filtration by program developed at JGI, and used for assembly Fieseler et al. (2004). including 454 reads (if available). For Illumina/454 hybrid assemblies, a combination of Velvet (Zerbino