Exo-Metabolome of Pseudovibrio sp. FO-BEG1 Analyzed by Ultra-High Resolution Mass Spectrometry and the Effect of Phosphate Limitation

Oceanic dissolved organic matter (DOM) is an assemblage of reduced carbon compounds, which results from biotic and abiotic processes. The biotic processes consist in either release or uptake of specific molecules by marine organisms. Heterotrophic bacteria have been mostly considered to influence the DOM composition by preferential uptake of certain compounds. However, they also secrete a variety of molecules depending on physiological state, environmental and growth conditions, but so far the full set of compounds secreted by these bacteria has never been investigated. In this study, we analyzed the exo-metabolome, metabolites secreted into the environment, of the heterotrophic marine bacterium Pseudovibrio sp. FO-BEG1 via ultra-high resolution mass spectrometry, comparing phosphate limited with phosphate surplus growth conditions. Bacteria belonging to the Pseudovibrio genus have been isolated worldwide, mainly from marine invertebrates and were described as metabolically versatile Alphaproteobacteria. We show that the exo-metabolome is unexpectedly large and diverse, consisting of hundreds of compounds that differ by their molecular formulae. It is characterized by a dynamic recycling of molecules, and it is drastically affected by the physiological state of the strain. Moreover, we show that phosphate limitation greatly influences both the amount and the composition of the secreted molecules. By assigning the detected masses to general chemical categories, we observed that under phosphate surplus conditions the secreted molecules were mainly peptides and highly unsaturated compounds. In contrast, under phosphate limitation the composition of the exo-metabolome changed during bacterial growth, showing an increase in highly unsaturated, phenolic, and polyphenolic compounds. Finally, we annotated the detected masses using multiple metabolite databases. These analyses suggested the presence of several masses analogue to masses of known bioactive compounds. However, the annotation was successful only for a minor part of the detected molecules, underlining the current gap in knowledge concerning the biosynthetic ability of marine heterotrophic bacteria.海洋溶解有机物（dissolved organic matter, DOM）是一类还原态碳化合物的集合体，由生物与非生物过程共同生成。其中生物过程涵盖海洋生物对特定分子的释放与摄取环节。以往研究多认为，异养细菌主要通过优先摄取部分化合物来改变DOM的组成。不过，这类细菌也会根据自身生理状态、环境条件与生长情况分泌多种分子，但迄今为止，尚未有研究对这类细菌分泌的全部化合物谱系开展系统性调查。本研究借助超高分辨率质谱技术，分析了异养海洋细菌假单胞弧菌属（Pseudovibrio）FO-BEG1菌株的胞外代谢组——即分泌至环境中的代谢物，对比了磷限制与磷充足两种生长条件下的差异。全球范围内均已分离得到假单胞弧菌属（Pseudovibrio）细菌，其宿主主要为海洋无脊椎动物，该类群被归类为代谢功能多样的α-变形菌纲（Alphaproteobacteria）成员。研究结果显示，该菌株的胞外代谢组规模与多样性远超预期，包含数百种分子式各异的化合物；其核心特征为动态的分子循环过程，且受菌株生理状态的显著影响。此外，磷限制会极大地改变分泌分子的总量与组成特征。通过将检测到的质荷比归为通用化学类别，本研究观察到：在磷充足条件下，分泌的分子主要为肽类与高度不饱和化合物；而在磷限制条件下，胞外代谢组的组成随细菌生长发生动态变化，高度不饱和、酚类及多酚类化合物的占比显著升高。最后，本研究依托多个代谢物数据库对检测到的质荷比进行注释。分析结果表明，存在多种与已知生物活性化合物质量数相似的质荷比信号，但仅少数检测到的分子得以成功注释，这凸显了当前学界在海洋异养细菌生物合成能力相关认知上的显著空白。