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 (AGMPFs). 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 high sensitivity, spatial resolution mass spectrometry and imaging 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 analysis of AP-SMALDI and cLSM imaging with resolution down to a single bacterial cell level can be easily applied to other microbial consortia and their hosts. The novelty of this contribution is the use of an in situ setup to resolve spatial distributions of metabolites and the identification of specific symbiotic bacteria, which is designed to avoid all types of external contamination.

共生作为海洋生态系统中一种常见的生命形式，已被多次观察到。例如，大型海藻与产生促进藻类生长和形态发生因子（AGMPFs）的细菌共存。在缺乏其必需的共生细菌Roseovarius sp. MS2和Maribacter sp. MS6的情况下，绿色大型海藻Ulva mutabilis（绿藻门）会发育成类似愈伤组织的表型。为了理解微生物在微观尺度上的相互作用，需要进行空间分辨研究。因此，我们采用了高灵敏度、空间分辨的质谱成像技术，以期对细菌与大型海藻之间的互利共生作用获得新的视角。通过大气压扫描显微镜辅助激光解吸/电离高分辨率质谱（AP-SMALDI-HRMS）和比较代谢组学，在Ulva的化学环境中鉴定出低分子量极性化合物。胆碱（2-羟基-N，N，N-三甲基乙烷-1-铵）仅在无菌条件下培养的藻类中检测到，而 ectoine（1，4，5，6-四氢-2-甲基-4-吡啶甲酸）则在细菌存在的情况下被发现。由于 ectoine 仅由这些细菌产生，因此被用作局部化研究的代谢标志物，以研究Roseovarius sp.在三分体群落中的定位。通过结合共聚焦激光扫描显微镜（cLSM）和AP-SMALDI-HRMS，我们证实了Roseovarius sp. MS2主要定居在U. mutabilis的根状区（固着器）中。我们的发现为在生态学相关背景下解码水生生态系统中多细胞宿主与细菌共生提供了基础。作为一种多功能的微生物组研究工具，将AP-SMALDI和cLSM成像与单个细菌细胞水平的分辨率相结合，可以轻松应用于其他微生物群落及其宿主。本贡献的革新之处在于使用原位装置解析代谢物的空间分布，并识别特定的共生细菌，该设计旨在避免所有类型的污染。