Single-cell genomics reveals complex carbohydrate degradation patterns in poribacterial symbionts of marine sponges

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 1 Department 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 Marine sponges (phylum Porifera) are the most ancient extant metazoans with fossil records, indicating their emergence more than 600 million years ago (Love et al., 2009). These animals are sessile filter feeders with an enormous filtering capacity that is known to affect nutrient concentrations in the surrounding environment (Gili and Coma, 1998; Maldonado et al., 2005, 2012). In addition to their evolutionary and ecological significance, sponges have attracted recent scientific attention owing to their specific and unique microbiology (Hentschel et al., 2012). The microbial biomass in sponges is located in the extracellular matrix, the so-called Correspondence: U Hentschel, Department of Botany II, Juliusvon-Sachs Institute for Biological Sciences, University of Wuerzburg, Julius-von-Sachs-Platz 3, Wuerzburg 97082, Germany. E-mail: Received 26 February 2013; revised 4 June 2013; accepted 4 June 2013; published online 11 July 2013 ‘mesohyl’, and can make up 35% of the sponge body mass (Vacelet, 1975). Collectively, representatives of more than 30 bacterial phyla and both archaeal lineages have so far been found in sponges from various geographic locations (Webster et al., 2010; Schmitt et al., 2012; Simister et al., 2012). The microbial diversity of marine sponges is well investigated (Taylor et al., 2007), and the collective repertoire of ‘omics’ approaches has been instrumental to shed light on the functional genomic traits of the collective sponge microbiome (Thomas et al., 2010; Fan et al., 2012; Liu et al., 2012; Radax et al., 2012). However, community-wide approaches do not provide sufficient information about functions of specific symbiont clades. Providing a thorough understanding of symbiont function is further complicated by the fact that many sponge symbiont lineages remain uncultivated, such as for the many candidate phyla found in these animals (Schmitt et al., 2012). One such candidate phylum, termed Candidate phylum Poribacteria, was originally discovered and Carbohydrate degradation in Poribacteria J Kamke et al 2288 described in marine sponges (Fieseler et al., 2004). Poribacteria are widely distributed and highly abundant in sponge species around the world (Fieseler et al., 2004; Lafi et al., 2009; Schmitt et al., 2011, 2012), and also occur freely in seawater, albeit at very low abundances (Pham et al., 2008; Webster et al., 2010; Taylor et al., 2013). They were shown to be affiliated with the Planctomycetes, Verrucomicrobia, Chlamydiae (PVC) superphylum (Wagner and Horn, 2006). Poribacteria are vertically transmitted via reproductive stages (Schmitt et al., 2008, Webster et al., 2010). Owing to their abundance and diversity within sponges, Poribacteria can be regarded as a model sponge symbiont. Single-cell genomics has become the most useful tool to investigate the genomic repertoire of distinct uncultivated microbial symbionts (Kamke et al., 2012) and other microorganisms (Woyke et al., 2009; Yoon et al., 2011; Stepanauskas, 2012). It also has previously been successfully applied to a poribacterial cell (Siegl et al., 2011). Unlike other ‘omics’ approaches, this method can connect phylogenetic identity with the functional potential of uncultivated microbial organisms, even from high diversity environments. Here we used single-cell genomics to analyze five poribacterial cells from the Mediterranean sponge Aplysina aerophoba, expanding the existing data set from one (Siegl et al., 2011) to a total of six poribacterial single-amplified genomes (SAGs). We provide an in-depth genomic analysis of one of the main symbiont lineages in the complex microbiota of marine sponges. The property of carbohydrate degradation emerged as the most common feature among the analyzed genomes. We therefore focused on carbohydrate degradation potential of Poribacteria in this study and discussed the results in context of a nutritional basis of the sponge–microbe symbiosis. Materials and methods Sample collection and processing Samples of the marine sponge A. aerophoba were collected in September 2009 by scuba diving to a depth of 5–12 m at the Coast of Rovinj, Croatia (451080 N, 131640 E). The animals were transported to the University of Wuerzburg (Wuerzburg, Germany) and kept in seawater aquaria until further processing within 1 week of collection. Fresh sponge samples were used for extraction of sponge-associated prokaryotes using an established protocol of tissue disruption, density centrif (...truncated)