Automatic profiling of lipids and lipopeptides from bacterial colonies by multimodal imaging with mass spectrometry and microscopy

Mass Spectrometry Imaging (MSI) is a method of growing interest for the in-situ study of lipopeptides and lipids produced during bacterial colony interactions on hard surfaces. However, this type of analysis is often limited to one interaction per MS image or very few interactions. With this practice, the heterogeneity of different bacteria, i.e., the diversity in molecular expression within the same bacterial strain, is not taken into account. Here, we propose a new informatic method that allows to study the molecular expression of multiple bacterial colonies from a single MS image by combining light microscopy and MALDI mass spectrometry imaging (FT-ICR). The method segments each colony on the different imaging modalities and estimates their signals. We have applied our method on an agar coculture of Bacillus velezensis GA1 and Pseudomonas sp. CMR12a. In this experiment, our method highlights a sub-cluster of CMR12a which is characterized by an over expression of particular PG lipids and an under expression of sessilins and orfamides. The colonies corresponding to this sub-cluster seem to be located closer to the interaction region with GA1 compared to the other CMR12a colonies. Further work will focus on in vivo analysis of bacteria colonizing plant’s roots.