The ISME Journal (2018) 12, 556–567 © 2018 International Society for Microbial Ecology All rights reserved 1751-7362/18 www.nature.com/ismej ORIGINAL ARTICLE In situ metabolomic- and transcriptomic-profiling of the host-associated cyanobacteria Prochloron and Acaryochloris marina Lars Behrendt1,2,3, Jean-Baptiste Raina4, Adrian Lutz5, Witold Kot6, Mads Albertsen7, Per Halkjær-Nielsen7, Søren J Sørensen3, Anthony WD Larkum4 and Michael Kühl2,4 1Department of Civil, Environmental and Geomatic Engineering, Swiss Federal Institute of Technology, Zürich, Switzerland; 2Marine Biological Section, Department of Biology, University of Copenhagen, Helsingør, Denmark; 3Microbiology Section, Department of Biology, University of Copenhagen, Copenhagen, Denmark; 4Plant Functional Biology and Climate Change Cluster (C3), University of Technology, Sydney, New South Wales, Australia; 5Metabolomics Australia, School of BioSciences, University of Melbourne, Parkville, Victoria, Australia; 6Department of Environmental Science—Enviromental Microbiology and Biotechnology, Aarhus University, Roskilde, Denmark and 7Center for Microbial Communities, Department of Chemistry and Bioscience, Aalborg University, Aalborg, Denmark The tropical ascidian Lissoclinum patella hosts two enigmatic cyanobacteria: (1) the photoendo- symbiont Prochloron spp., a producer of valuable bioactive compounds and (2) the chlorophyll-d containing Acaryochloris spp., residing in the near-infrared enriched underside of the animal. Despite numerous efforts, Prochloron remains uncultivable, restricting the investigation of its biochemical potential to cultivation-independent techniques. Likewise, in both cyanobacteria, universally important parameters on light-niche adaptation and in situ photosynthetic regulation are unknown. Here we used genome sequencing, transcriptomics and metabolomics to investigate the symbiotic linkage between host and photoendosymbiont and simultaneously probed the transcriptional response of Acaryochloris in situ. During high light, both cyanobacteria downregulate CO2 fixing pathways, likely a result of O2 photorespiration on the functioning of RuBisCO, and employ a variety of stress-quenching mechanisms, even under less stressful far-red light (Acaryochloris). Metabo- lomics reveals a distinct biochemical modulation between Prochloron and L. patella, including noon/ midnight-dependent signatures of amino acids, nitrogenous waste products and primary photo- synthates. Surprisingly, Prochloron constitutively expressed genes coding for patellamides, that is, cyclic peptides of great pharmaceutical value, with yet unknown ecological significance. Together these findings shed further light on far-red-driven photosynthesis in natural consortia, the interplay of Prochloron and its ascidian partner in a model chordate photosymbiosis and the uncultivability of Prochloron. The ISME Journal (2018) 12, 556–567; doi:10.1038/ismej.2017.192; published online 31 October 2017 Introduction aspects of this obligate symbiosis have been resolved, for example, it is known that Prochloron Colonial ascidians (family didemnidae) are the only contributes to carbon fixation/nutrient translocation chordates known to form an obligate photosymbiosis within its host (Pardy and Lewin, 1981; Kremer (Hirose et al., 2009) with the chorophyll (Chl) et al., 1982; Griffiths and Thinh, 1983) and produces b-containing cyanobacterium Prochloron spp. bioactive secondary metabolites such as patella- (Lewin and Withers, 1975). Prochloron is found at mides (Schmidt et al., 2005). Other physiological high cell densities in the cloacal cavity ( = CC) of aspects, such as N2 fixation (Kline and Lewin, 1999), some didemnid species, or more rarely, as epibionts reactive-oxygen species detoxification (Lesser and (Hirose et al., 2009; Nielsen et al., 2015). Certain Stochaj, 1990), host interaction and the diel regula- tion of metabolic pathways have remained largely Correspondence: L Behrendt, Department of Civil, Environmental unstudied, mostly due to the inability to maintain and Geomatic Engineering, Swiss Federal Institute of Technology, cultures of Prochloron. Culture-independent meth- Stefano-Franscini Platz 5, HIL G37.1, CH-8093 Zurich, ods have elucidated certain aspects of Prochloron Switzerland. biology, and the publication of a draft-genome E-mail: [email protected] Received 8 July 2017; revised 25 September 2017; accepted 26 (Donia et al., 2011) allowed insights into the September 2017; published online 31 October 2017 functional ecology of this symbiont. This first In situ metabolomic- and transcriptomic-profiling L Behrendt et al 557 genomic analysis (Donia et al., 2011) demonstrated depth during low tide (~2.8 m tidal range). Collected that Prochloron carries all primary metabolic genes specimens were kept in an outdoor aquarium (max. required for survival outside of its host, along with a ~ 200 μmol photons m − 2 s − 1) with a continuous large amount of paralogous high-light inducible supply of fresh seawater (26–28 °C). Sampling of L. genes (Hli). Additionally, Prochloron has genes patella-associated microbial biofilms for transcrip- coding for the nitrate reduction pathway but appears tomics was performed during night-time to lack the capability to fix dinitrogen (Donia et al., (0000–0300 hours, hereafter referred to as ‘mid- 2011), contrasting previous reports that demon- night’) or during midday (1200–1400 hours, ‘ ’ strated active N2-fixation (Kline and Lewin, 1999). hereafter referred to as noon ) over 2 consecutive Prochloron is the most abundant bacterium within days (14–16.02.2011). During noon, photon irra- the didemnid ascidian Lissoclinum patella diances at the sampling site routinely reached (Behrendt et al., 2012) but other bacteria co-occur 41500 μmol photons m2 s − 1. Sampling for metabolo- in the CC, the surface and the underside (US) of the mic analysis was done for a single diel cycle during a animal (Behrendt et al., 2012). One of these bacteria different field campaign (07–18.02.2017) using L. is Acaryochloris marina, a Chl d-containing cyano- patella specimens from the same sampling site and bacterium residing below Prochloron on the US of water depth. Sampling was done with a sharpened the host (Kühl et al., 2005; Behrendt et al., 2012). In cork-borer (10 mm diameter), inserted into live L. this particular microenvironment, Acaryochloris patella specimen. The resulting core was sub- uses Chl d to sustain oxygenic photosynthesis under sampled into two microbial consortia: the CC, near-infrared radiation, that propagates through the containing the internal symbiont Prochloron and overlying ascidian tissue and Prochloron cells which the biofilm associated with the US of L. patella selectively filter out visible light (Kühl et al., 2005; (Figure 1a and Behrendt et al., 2012). For transcrip- Behrendt et al., 2012). Acaryochloris is a significant tomics, three subsamples were pooled and flash- member of the microbial community found on the frozen in liquid N2. Three biological replicates were US of L. patella, accounting for up to 14% of all sampled for each time point and day. For genomic sequences (Behrendt et al., 2012). Because it con- sequencing, Prochloron cells were collected via a tains Chl d and is easily cultivated, Acaryochloris Pasteur pipette inserted through the siphon into the has been studied extensively; yet little is known CC of L. patella. The resulting cells were filtered about its in situ ecology and functional niche through meshes (100 μm and 50 μm) and concen- adaptation mechanisms. trated using centrifugation (2000 g for 2 min). The Most information about the diversity and physiol- supernatant was removed and Prochloron cells were ogy of Prochloron and Acaryochloris has been flash frozen in liquid N2. For metabolomics, 10 obtained through metagenomics and amplicon biological replicates were sampled in a randomized sequencing (Donia et al., 2011; López-Legentil fashion from four individual animals and the CC et al., 2011; Behrendt et al., 2012), and a few studies cores were snap frozen in liquid N2 during the day on in vitro gene expression of Acaryochloris and night. All samples were transported back to the (Pfreundt et al., 2012; Yoneda et al., 2016). Here laboratory on dry ice and stored at − 80 °C upon we used transcriptomics on in vivo microbial arrival. communities within the CC and the US of L. patella to obtain insights to the regulatory pathways employed by Prochloron and Acaryochloris during Prochloron genome sequencing the noon and midnight. For the assessment of in situ Frozen Prochloron cells were homogenized in liquid transcripts, we used the published A. marina N2 in a bleach-cleaned mortar. The resulting powder MBIC11017 genome (Swingley et al., 2008) and was processed using the standard FastDNA for soil generated a new draft genome of Prochloron ( = P5), kit (MP Biomedicals, Illkirch Cedex, France) with using refined assembly methods (Albertsen et al., two additional bead-beating cycles. The resulting 2013). Moreover, metabolomics was used on the CC DNA was eluted in TAE buffer and quantified using to assess the diel biochemical modulation between a Qubit-fluorometer (Invitrogen, San diego, CA, Prochloron and its host. The results are discussed USA), checked for integrity on a 0.8% agarose gel against the background of the microenvironment of and stored at − 20 °C. Sequencing was performed Prochloron and Acaryochloris (Behrendt et al., 2012; using Illumina technology (Illumina Inc., San diego, Kühl et al., 2012) and in