The ISME Journal (2021) 15:3062–3075 https://doi.org/10.1038/s41396-021-00987-x ARTICLE Isotopic tracing reveals single-cell assimilation of a macroalgal polysaccharide by a few marine Flavobacteria and Gammaproteobacteria 1 1 2,3 4 5 6 François Thomas ● Nolwen Le Duff ● Ting-Di Wu ● Aurélie Cébron ● Stéphane Uroz ● Pascal Riera ● 7 8 8 2,3 Cédric Leroux ● Gwenn Tanguy ● Erwan Legeay ● Jean-Luc Guerquin-Kern Received: 6 January 2021 / Revised: 25 March 2021 / Accepted: 9 April 2021 / Published online: 5 May 2021 © The Author(s) 2021. This article is published with open access Abstract Algal polysaccharides constitute a diverse and abundant reservoir of organic matter for marine heterotrophic bacteria, central to the oceanic carbon cycle. We investigated the uptake of alginate, a major brown macroalgal polysaccharide, by microbial communities from kelp-dominated coastal habitats. Congruent with cell growth and rapid substrate utilization, alginate amendments induced a decrease in bacterial diversity and a marked compositional shift towards copiotrophic bacteria. We traced 13C derived from alginate into specific bacterial incorporators and quantified the uptake activity at the single-cell level, 1234567890();,: 1234567890();,: using halogen in situ hybridization coupled to nanoscale secondary ion mass spectrometry (HISH-SIMS) and DNA stable isotope probing (DNA-SIP). Cell-specific alginate uptake was observed for Gammaproteobacteria and Flavobacteriales, with carbon assimilation rates ranging from 0.14 to 27.50 fg C µm−3 h−1. DNA-SIP revealed that only a few initially rare Flavobacteriaceae and Alteromonadales taxa incorporated 13C from alginate into their biomass, accounting for most of the carbon assimilation based on bulk isotopic measurements. Functional screening of metagenomic libraries gave insights into the genes of alginolytic Alteromonadales active in situ. These results highlight the high degree of niche specialization in heterotrophic communities and help constraining the quantitative role of polysaccharide-degrading bacteria in coastal ecosystems. Introduction Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41396- 021-00987-x. By recycling a large proportion of the available organic matter, heterotrophic bacteria control the oceanic fluxes of * François Thomas carbon and energy [1]. The dissolved fraction is the largest [email protected] reservoir of oceanic organic matter, with a global estimate of 1 Sorbonne Université, CNRS, Integrative Biology of Marine 662 Pg dissolved organic carbon (DOC) [2, 3]. Marine DOC Models (LBI2M), Station Biologique de Roscoff (SBR), comprises diverse low- and high-molecular-weight com- Roscoff, France pounds, of which carbohydrates constitute 15–50% [4], 2 Institut Curie, Université Paris-Saclay, Paris, France mostly derived from micro- and macro-algae in the surface 3 Université Paris-Saclay, INSERM US43, CNRS UMS2016, ocean. With an estimated standing stock amounting hun- Multimodal Imaging Center, Orsay, France dreds of megatons in temperate and sub-polar habitats [5, 6], 4 Université de Lorraine, CNRS, LIEC, Nancy, France brown macroalgae are major primary producers in coastal 5 Université de Lorraine, INRAE, UMR1136 « Interactions Arbres- regions. Brown macroalgae of the order Laminariales, col- Microorganismes », Champenoux, France lectively known as kelps, can release up to 25% of the fixed −2 −1 6 Sorbonne Université, CNRS, UMR7144, Station Biologique de carbon as exudates, thus contributing ~1.3 kg C m y to Roscoff (SBR), Roscoff, France the DOC pool [7, 8]. This significantly impacts coastal 7 CNRS, Sorbonne Université, FR2424, Metabomer, Station ecosystems, where kelp forests locally increase DOC con- Biologique de Roscoff, Roscoff, France centrations and sustain distinct microbial communities [9]. – −1 8 CNRS, Sorbonne Université, FR2424, Genomer, Station Previous studies measured 1 1.5 mg C L as DOC in sea- Biologique de Roscoff, Roscoff, France water from kelp beds [9, 10]. Kelp exudates contain simple Isotopic tracing reveals single-cell assimilation of a macroalgal polysaccharide by a few marine. 3063 sugars, polysaccharides, proteins, lipids, and aromatic (HISH-SIMS) to trace 13C-labeled alginate into specific compounds [11, 12]. In particular, the polysaccharide algi- incorporators within a coastal seawater community and nate, which comprises ca. half of kelp dry biomass [13], quantify the uptake activity at the single-cell level. We show accounts for 5% of the released DOC [11]. Given the density that most of the alginate is used by a few initially rare and high primary productivity of kelps in coastal regions, Flavobacteriaceae and Gammaproteobacteria taxa and alginate therefore constitutes an abundant resource for provide the first single-cell estimates of alginate incorpora- planktonic bacteria, available year-round but with potential tion rates. seasonal variations in quantities [7]. Alginate is a linear polysaccharide consisting of β-D-mannuronic acid and α-L- guluronic acid arranged in homo- or hetero-polymeric Methods blocks and with varying proportions of each motif depend- ing on algal species, tissue, age, and seasons [14, 15]. Many Alginate production isolated marine bacteria can degrade alginate and use it as a carbon and energy source. Alginolytic culturable repre- Natural (12C-natural) and 13C-enriched alginates were pre- sentatives belong to Proteobacteria, Flavobacteriales, Fir- pared as described previously [40]. Briefly, the kelp Lami- micutes, and Verrucomicrobiae [16–19]. In particular, naria digitata was cultivated with either natural (98.9 12C%) 13 13 several polysaccharide utilization loci (PUL) dedicated to or C-labeled (99 C%) NaHCO3. Alginate was chemically alginate assimilation have been described, which encode extracted as detailed in Supplementary Methods. The average alginate lyases for the breakdown of the substrate into oli- molecular weight of alginate estimated using multi-angle gosaccharides, SusC/SusD homologs for binding and uptake laser light scattering (MALLS) was Mw = 1.7 105 Da. of degradation products, and cytoplasmic enzymes proces- Alginates were assayed for residual proteins and DNA using sing monomers into the central metabolism [20–23]. Algi- the Qubit Protein Assay kit and dsDNA HS assay kit nolytic PULs are widespread in marine Flavobacteriaceae (ThermoFisher Scientific), respectively. 12C-natural alginate and were transferred to Gammaproteobacteria [20]. Their contained 13.7 mg protein and 284 µg DNA g−1 (i.e., 98.6% expression is tightly controlled by PUL-encoded regulators, purity). 13C-enriched alginate contained 10.1 mg protein and allowing a rapid and massive overexpression when alginate 288 µg DNA g−1 (i.e., 99.0% purity). The absence of inhi- becomes available, and might be fine-tuned by yet unknown bitors was confirmed by checking the growth of the algino- cross-regulation, catabolic repression, or substrate prior- lytic strain Zobellia galactanivorans DsijT in minimum itization mechanisms [24–26]. Depending on the type and medium supplemented with 12C-natural or 13C-enriched cellular localization of their alginate lyases, marine bacteria alginate (1 g l−1). Alginate isotopic ratio was measured by adopt different ecophysiological strategies leading to niche elemental analysis coupled to isotope ratio mass spectro- specialization towards polymeric alginate vs. oligomeric metry (EA-IRMS) as described previously [40]. 12C-natural products [27, 28]. Despite the characterization of alginate and 13C-enriched alginate had 1.1337 ± 0.0004 and 3.6279 ± catabolism in cultivated strains, identifying the members of 0.0009 13C atom percent, respectively (mean ± s.d. n = 3 natural bacterial communities that use alginate remains a technical replicates). Before microcosm amendment, algi- challenge. Previous studies showed that alginate amend- nates were solubilized (5 g l−1)inTris-HCl50mMpH8.0 ments induce shifts in seawater community composition and autoclaved. The average molecular weight of autoclaved [29–32] and provided insights into the global community alginate determined by MALLS analysis was Mw = 1.4 105 response and alginolytic potential. However, these approa- Da with a polydispersity index of 1.364, corresponding to an ches cannot directly link alginate utilization to individual average degree of polymerization of ca. 800 monomers. bacterial taxa nor estimate substrate assimilation at the single-cell level, hindering our comprehension of alginate Seawater microcosms fate in marine ecosystems. To date, few studies focused on the direct detection and quantification of the utilization of a Natural seawater was collected at 12.30 pm on January 21 given substrate by individual bacteria in natural samples, 2019 from a kelp-dominated tidal pool at Le Bloscon, using e.g., microautoradiography, Raman spectroscopy, Roscoff, France (48°43'33.18“N, 3°58'7.58“W). Average fluorescently-labeled substrates, or stable isotope probing surface water temperature, salinity, and pH are 10–12 °C, (SIP) [33, 34]. To our knowledge, SIP has seldom been 35.0–35.2 g kg−1,and 7.75–7.80 in January in this area applied using complex marine organic matter [35–39], and (values from long-term monitoring SOMLIT Estacade Sta- never to macroalgae-derived polysaccharides, despite their tion, 1.2 km from sampling site). Autoclaved plastic car- importance in the carbon cycle. Here, we combined DNA- boys were rinsed three times with water from the site before SIP with halogen in