Search

Nanospray desorption electrospray ionization mass spectrometry, a powerful ambient sampling and imaging technique, is herein coupled as an isolated source with 21 Tesla (21T) Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS). Absorption-mode data, enabled by an external data acquisition system, is applied for improved mass resolution, accuracy, and dynamic range without compromising spectral acquisition rates. Isotopic fine structure (IFS) information is obtained from the ambient sampling of living Bacillusand Fusariumspecies, allowing for high confidence in molecular annotations with a resolution >830 k (at m/z825). Tandem mass spectrometry experiments for biological samples are shown to retain the IFS in addition to gained fragmentation information, providing a further degree of annotation confidence from ambient analyses. Rat brain was imaged by nanospray desorption electrospray ionization (nano-DESI) 21T FTICR MS in ∼5 h using 768 ms transients, producing over 800 molecular annotations using the METASPACE platform and low-parts-per-billion mass accuracy at a spatial resolution of ∼25 × 180 μm. Finally, nano-DESI 21T FTICR MS imaging is demonstrated to reveal images corresponding to the IFS, as well as hundreds of additional molecular features (including demonstrated differences as low as 8.96 mDa) that are otherwise undetected by a more conventional imaging methodology.

Microbiomes play critical roles in ecosystems and human health, yet in most cases scientists lack standardized and reproducible model microbial communities. The development of fabricated microbial ecosystems, which we term EcoFABs, will provide such model systems for microbiome studies.

ABSTRACTThere is a growing appreciation for the impact that bacteria have on higher organisms. Plant roots often harbor beneficial microbes, such as the Gram-positive rhizobacterium Bacillus subtilis, that influence their growth and susceptibility to disease. The ability to form surface-attached microbial communities called biofilms is crucial for the ability of B. subtilisto adhere to and protect plant roots. In this study, strains harboring deletions of the B. subtilisgenes known to synthesize and degrade the second messenger cyclic di-adenylate monophosphate (c-di-AMP) were examined for their involvement in biofilm formation and plant attachment. We found that intracellular production of c-di-AMP impacts colony biofilm architecture, biofilm gene expression, and plant attachment in B. subtilis. We also show that B. subtilissecretes c-di-AMP and that putative c-di-AMP transporters impact biofilm formation and plant root colonization. Taken together, our data describe a new role for c-di-AMP as a chemical signal that affects important cellular processes in the environmentally and agriculturally important soil bacterium B. subtilis. These results suggest that the “intracellular” signaling molecule c-di-AMP may also play a previously unappreciated role in interbacterial cell-cell communication within plant microbiomes.IMPORTANCEPlants harbor bacterial communities on their roots that can significantly impact their growth and pathogen resistance. In most cases, however, the signals that mediate host-microbe and microbe-microbe interactions within these communities are unknown. A detailed understanding of these interaction mechanisms could facilitate the manipulation of these communities for agricultural or environmental purposes. Bacillus subtilisis a plant-growth-promoting bacterium that adheres to roots by forming biofilms. We therefore began by exploring signals that might impact its biofilm formation. We found that B. subtilissecretes c-di-AMP and that the ability to produce, degrade, or transport cyclic di-adenylate monophosphate (c-di-AMP; a common bacterial second messenger) affects B. subtilisbiofilm gene expression and plant attachment. To our knowledge, this is the first demonstration of c-di-AMP impacting a mutualist host-microbe association and suggests that c-di-AMP may function as a previously unappreciated extracellular signal able to mediate interactions within plant microbiomes.