A new glance at the chemosphere of macroalgal-bacterial interactions: In situ profiling of metabolites in symbiosis by mass spectrometry.

Symbiosis is a dominant form of life that has been observed numerous times in marine ecosystems. For example, macroalgae coexist with bacteria that produce factors that promote algal growth and morphogenesis. The green macroalga Ulva mutabilis (Chlorophyta) develops into a callus-like phenotype in the absence of its essential bacterial symbionts Roseovarius sp. MS2 and Maribacter sp. MS6. Spatially resolved studies are required to understand symbiont interactions at the microscale level. Therefore, we used mass spectrometry profiling and imaging techniques with high spatial resolution and sensitivity to gain a new perspective on the mutualistic interactions between bacteria and macroalgae. Using atmospheric pressure scanning microprobe matrix-assisted laser desorption/ionisation high-resolution mass spectrometry (AP-SMALDI-HRMS), low-molecular-weight polar compounds were identified by comparative metabolomics in the chemosphere of Ulva. Choline (2-hydroxy-N,N,N-trimethylethan-1-aminium) was only determined in the alga grown under axenic conditions, whereas ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) was found in bacterial presence. Ectoine was used as a metabolic marker for localisation studies of Roseovarius sp. within the tripartite community because it was produced exclusively by these bacteria. By combining confocal laser scanning microscopy (cLSM) and AP-SMALDI-HRMS, we proved that Roseovarius sp. MS2 settled mainly in the rhizoidal zone (holdfast) of U. mutabilis. Our findings provide the fundament to decipher bacterial symbioses with multicellular hosts in aquatic ecosystems in an ecologically relevant context. As a versatile tool for microbiome research, the combined AP-SMALDI and cLSM imaging analysis with a resolution to level of a single bacterial cell can be easily applied to other microbial consortia and their hosts. The novelty of this contribution is the use of an in situ setup designed to avoid all types of external contamination and interferences while resolving spatial distributions of metabolites and identifying specific symbiotic bacteria.