MALDI Mass Spectrometry Imaging: a new tool to decipher the antibiome of Bacillus amyloliquefaciens

Soil Bacillus isolates may devote up to 8% of their genome to nonribosomal synthesis of lipopeptide (LP)- and polyketide (PK)-type antibiotics. LPs from surfactin, iturin and fengycin families are known to exert different actions on the wellness of the producing strain such as fungitoxicity (iturin, fengycin) or motility, root colonization and immune stimulating agent (surfactin). Nevertheless, few is reported about the actual antibiome secreted in situ by Bacillus cells during confrontation with phytopathogens or plant root colonization.
We developed a method mimicking the conditions prevailing in the rhizosphere and, taking advantage of the versatility of Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging, we were able to localize and identify antibiotics produced in situ by bacterial cells.
First, we applied this new methodology to bioassays in which Bacillus amyloliquefaciens 98S were grown together with Fusarium oxysporum, with the aim of deciphering the role of the different LP families during the phytopathogen growth inhibition. Our results showed that the three LP families were readily produced in different proportions. Especially, images of surfactins, iturins and fengycins demonstrated that iturins are the antibiotic family actually involved in the antagonism against Fusarium oxysporum.
In a second approach, we used a “in planta” model in which Bacillus amyloliquefaciens S499 was simultaneously grown with Tomato and Arabidopsis thaliana roots. Imaging results, obtained during a time course analysis, showed that surfactin is always the major lipopeptide detected. In further experiments involving a refined time-window, we observed that surfactin is actually produced as soon as 24h post inoculation. These results were the starting point of a wider study showing that the early accumulation of surfactin is a complex phenomenon involving, among other mechanisms, cell-well components recognition by bacteria, and that this interaction is a win-win association for both plant and bacterial cells.