Abstract
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.
Highlights
In intertidal zones with high temporal and spatial ecosystem variations, bacteria and macroalgae establish close mutualistic relationships, in which both gain reciprocal benefits forming an ecological unit [1,2,3]
The algal germlings incubated with the marine bacteria showed a rhizoidal zone that serves for substrate attachment and a thallus zone
Metabolic profiling of whole alga and specialised tissues conducted with AP-SMALDI-HRMS enabled identifying specific metabolites in host–bacteria symbiosis
Summary
In intertidal zones with high temporal and spatial ecosystem variations, bacteria and macroalgae establish close mutualistic relationships, in which both gain reciprocal benefits forming an ecological unit (holobiont) [1,2,3]. Comparative metabolomics using atmospheric pressure scanning microprobe matrix-assisted laser desorption/ ionisation high-resolution mass spectrometry (AP-SMALDIHRMS) enables the identification of specialised metabolites of the marine macroalga Ulva mutabilis (Chlorophyta) and its associated essential bacteria, a model system for cross-kingdom interactions [7]. The metabolic profiles of tissue and whole alga were obtained from callus or alga in symbiosis using AP-SMALDI-HRMS with the two sample preparations, either with matrix deposition (MALDIHRMS) or matrix-free analysis (LDI-HRMS) (Figure 1a and b).
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