Environmental Microbiology (2019) 00(00), 00–00 doi:10.1111/1462-2920.14560 Microdiversity and temporal dynamics of marine bacterial dimethylsulfoniopropionate genes Brent Nowinski,1 Jessie Motard-Côté,2,3 co-occurred with haptophytes. This in situ study of Marine Landa,1† Christina M. Preston,4 the drivers of DMSP fate in a coastal ecosystem Christopher A. Scholin,4 James M. Birch,4 demonstrates for the first time correlations between Ronald P. Kiene2,3 and Mary Ann Moran 1* specific groups of bacterial DMSP degraders and 1Department of Marine Sciences, University of Georgia, phytoplankton taxa. Athens, GA, 30602, USA. 2 Department of Marine Sciences, University of South Introduction Alabama, Mobile, AL, 36688, USA. 3Dauphin Island Sea Lab, Dauphin Island, AL, The phytoplankton-produced organic sulfur compound 36528, USA. dimethylsulfoniopropionate (DMSP) plays a major role in 4Monterey Bay Aquarium Research Institute, Moss marine microbial food webs as a source of reduced sulfur Landing, CA, 95039, USA. and carbon (Kiene et al., 2000), and its degradation has impacts on ocean–atmosphere sulfur flux (Andreae, 1990). Marine bacteria can catabolize DMSP using the Summary demethylation pathway, wherein sulfur is routed to metha- Dimethylsulfoniopropionate (DMSP) is an abundant nethiol and potentially incorporated into biomass; or using organic sulfur metabolite produced by many phyto- the cleavage pathway, in which case sulfur is routed to the plankton species and degraded by bacteria via two dis- gas dimethylsulfide (DMS) with implications for atmo- tinct pathways with climate-relevant implications. We spheric aerosol dynamics and cloud formation (Charlson assessed the diversity and abundance of bacteria pos- et al., 1987; Quinn and Bates, 2011). The gene dmdA sessing these pathways in the context of phytoplank- catalyses the first step in the demethylation pathway, ton community composition over a 3-week time period removing a methyl group and yielding methylmercaptopro- spanning September–October, 2014 in Monterey Bay, pionate, while seven different non-homologous ddd genes CA. The dmdA gene from the DMSP demethylation can catalyse the first step in cleavage, generating DMSP pathway dominated the DMSP gene pool and was har- and acrylate (Howard et al., 2006; Curson et al., 2008; boured mostly by members of the alphaproteobacterial Todd et al., 2009; 2011; 2012; Peng et al., 2012; Sun SAR11 clade and secondarily by the Roseobacter et al., 2016). dmdA and the ddd genes can be considered group, particularly during the second half of the study. gatekeeper genes for the entry of DMSP into these two Novel members of the DMSP-degrading community competing pathways. emerged from dmdA sequences recovered from meta- The main taxa implicated in most DMSP degradation genome assemblies and single-cell sequencing, includ- studies are marine members of the class Alphaproteo- ing largely uncharacterized gammaproteobacteria and bacteria, with genes discovered and characterized in the alphaproteobacteria taxa. In the DMSP cleavage path- Roseobacter group, the SAR11 clade and the SAR116 way, the SAR11 gene dddK was the most abundant early in the study, but was supplanted by dddP over cluster. The first bacterial dmdA gene described was in a time. SAR11 members, especially those harbouring model coastal heterotrophic bacterium from the Roseo- genes for both DMSP degradation pathways, had a bacter group, Ruegeria pomeroyi (Howard et al., 2006), strong positive relationship with the abundance of dino- and was subsequently found in many other roseobacters. flagellates, and DMSP-degrading gammaproteobacteria Many members of this group also harbour DMSP cleav- age genes dddD, dddL, dddP, dddQ and dddW, either Received 17 November, 2018; revised Month 2019; accepted 9 Feb- alone or in various combinations (Moran et al., 2012). ruary, 2019. *For correspondence. E-mail [email protected]; Tel. Many SAR11 bacteria also possess dmdA (Howard 706-542-6481; Fax 706-542-5888. †Present address: Ocean Sci- ences Department, University of California, Santa Cruz, CA et al., 2006), and recent research has discovered dddK, 95064, USA. a new DMSP cleavage gene specific to SAR11 cells © 2019 Society for Applied Microbiology and John Wiley & Sons Ltd. 2 B. Nowinski et al. (Sun et al., 2016). SAR116 members can possess dmdA picoeukaryotes from the Mamiellophyceae, diatoms from and dddP (Oh et al., 2010). the Bacillariophyceae and pelagophytes (Pelagophyceae) The coastal upwelling system of Monterey Bay, CA, increasing in relative abundance (Fig. 1B and C), coinci- harbours diverse and rapidly changing phytoplankton dent with the lowest chlorophyll a and DMSPt concentra- communities and high numbers of DMSP-degrading bac- tions measured during the study period (Fig. 1A). The teria (Varaljay et al., 2015). How these shifting phyto- bacterial community was dominated by Alphaproteobac- plankton communities, and thus, DMSP availability, may teria, including members of the known DMSP-catabolizing affect the DMSP-degrading bacterial community and the groups SAR11, Roseobacter and SAR116. Metagenomic metabolic routing of organic sulfur to the divergent bio- reads mapping to Gammaproteobacteria and Bacteroi- geochemical fates of the two degradation pathways were detes were also highly represented (Fig. 1D). There was a the questions that drove our study. We utilized the envi- significant negative linear regression of the relative abun- ronmental sample processor (ESP), an autonomous dance over time for SAR11 taxa (R2 =0.65;p <0.01). robotic instrument than can remain deployed in an envi- ronment for an extended period to repeatedly collect and Demethylation gene diversity preserve microbial community samples for later nucleic acid sequencing (Ottesen et al., 2011). The ESP sam- To characterize the phylogeny of predicted dmdA genes pled surface waters in Monterey Bay for 3 weeks in the in the metagenomic data, a base tree for read placement fall of 2014. Six sample dates from this collection varying was constructed with full-length DmdA sequences in DMSP and chlorophyll a levels were selected for meta- obtained by three methods: (1) assembled from the meta- genomic analysis of DMSP-related genes. We examined genomes using two different approaches (see Methods), the diversity and abundance of the DMSP-degrading bac- (2) obtained from single amplified genomes (SAGs) from terial community using metagenomic and single cell cells collected at the ESP deployment site and sequencing in the context of phytoplankton dynamics and (3) acquired from the NCBI RefSeq database based on environmental parameters. best hits to the metagenomic reads (Fig. 2; See Methods). For approach 2, six single cell bacterial genomes from seawater collected at the ESP mooring Results were selected for sequencing following screening for 16S rRNA genes indicative of likely DMSP-degrading taxa; Overview of microbial dynamics these included three from the SAR11 clade and three from Concentrations of chlorophyll a (as a proxy for phytoplank- the Roseobacter group (see Methods). After building the ton biomass) and total dissolved plus particulate DMSP dmdA base tree, metagenomic reads identified as dmdA concentration (DMSPt) each varied about 10-fold during the based on BLAST analysis against a custom reference data- − course of the study, from 0.8 to 7.2 ug L 1 chlorophyll a and base were placed on the tree and relative abundances from 32 to 311 nM DMSPt (Fig. 1A). Based on metage- were calculated by normalizing to the universal single copy nomic data, the phytoplankton community was dominated bacterial gene recA (Howard et al., 2006). by reads mapping to diatoms (Coscinodiscophyceae, Bacil- From this approach, it emerged that SAR11 was the lariophyceae, Mediophyceae and Fragilariophyceae), hapto- most abundant DMSP-demethylating taxon, with one in five phytes (Haptophyceae), picoeukaryotes (Mamiellophyceae) bacterial cells in Monterey Bay possessing a SAR11-like and dinoflagellates (Dinophyceae; Fig. 1B and C), with larg- dmdA (Fig. 2). Most SAR11 dmdA reads aligned with the est changes in relative abundance occurring towards the SAR11 subclade containing strain HTCC9022, and this end of the deployment. Centric diatoms from the Coscino- taxon recruited the most dmdA reads during the study over- discophyceae and dinoflagellates peaked in relative abun- all. Also abundant were dmdA readsmappingtoaSAR11 dance early in the study (September 29 sample date, subclade represented by Monterey Bay SAG SCGC-AG- corresponding to the peak of DMSPt concentration). 145-C19 obtained in this study. dmdA reads mapping to Pennate diatoms from the Bacillariophyceae exhibited a reference genomes from SAR11 subclade 1a.1, a group marked increase in relative abundance in the second typical of temperate coastal regions (Brown et al., 2012; week in October (October 8 and 13 sample dates). Hapto- Giovannoni, 2017), including HTCC1062 and HTCC1002, phytes showed highest relative abundances during the represented the third most abundant SAR11 DmdA group. first 2 weeks of the deployment (September 22, 29, The subclade including HTCC7211, which more typically October 3, 6 sample dates). There was a significant nega- dominates in warm, stratified open oceans, was not abun- tive linear regression of relative abundance over time for dant (Fig. 2). Haptophyceae (R2 =0.35;p < 0.05) and Dinophyceae DmdA sequences mapping to roseobacters made up (R2 =0.51;p < 0.01). At the October 13 sample date, the the next most frequent group. The sequence from