Introduction
Bacillus subtilis is a Gram-positive, spore-forming soil bacterium known for its ability to form robust, yet adaptive biofilms on various surfaces. It serves as an important model organism in microbiological research. To improve our understanding about the complex communication and cell differentiation processes occurring during biofilm formation, MALDI-2 mass spectrometry imaging (MSI) can be used. Here we advanced this method towards the analysis of thin sagittal sections across bacterial biofilms at 5-10 µm pixel size and for the top-view analysis of single and multiple B. subtilis knock-outs that are lacking cannibalism-associated peptide toxins and defense strategies.
Experimental
B. subtilis biofilms of wild type NCIB 3610 and NCIB 3610-derived mutant strains were grown at 28 °C for up to 10 d using a mixed cellulose ester filter membrane (mean pore size, 0.22 µm) as substrate, placed on minimal medium (MSgg) agar. For cryo-sectioning at 10 µm thickness, samples were embedded in carboxymethyl cellulose gel; whole biofilms were analyzed without embedding but following chemical fixation in 10% paraformaldehyde for 30 min. For MALDI analysis, all samples were spray- coated with 2,5-dihydroxyacetophenone MALDI matrix. A timsTOF fleX MALDI-2 mass spectrometer (Bruker Daltonics) was used as mass analyzer. Bright-field and phase contrast images were recorded with a VS200 microscopy scanner (Evident).
Results
In the course of evolution, B. subtilis has developed sophisticated strategies of cell-to-cell communication and division of labor to orchestrate colony growth in response to the environment, as for example availability of nutrients and presence of competing microorganisms. Cannibalism, a scenario in which a sub-population of cells is sacrificed to ensure colony survival, is for instance controlled via the expression of a set of peptide toxins. In particular, the roles of the epipeptide EPE, the sporulation killing factor SKF and the sporulation delaying peptide SDP, comprising 17, 26, and 42 AA, respectively, are currently studied to better understand these processes. Our MALDI-(2-)MSI analysis of cross-sections of B. subtilis wt strains enabled visualization of these peptides, exhibiting molecular masses between 2.1 and 4.3 kDa, at a spatial resolution in the low 10 µm-range. In addition, B. subtilis lipopeptides surfactins and plipastatins, as well as numerous further analytes in the m/z range below 1000 were detected (e.g. structural phospholipids) following adjustments in the matrix coating protocol. Confirmed by high-resolving microscopy, the intricate structures constituting the biofilms, including crypt and microchannel formation, became observable on a molecular level. Our MALDI-MSI analysis of single and multiple knockouts of the epe, sdp and skf operons, in this case obtained solely from whole matrix-coated biofilms, added yet another biological and biochemical dimension to these findings. For example, our experiments visualized distinct “ wave-like” expression of the toxins across the biofilm.
Together, our mass spectrometry imaging data reflect an exceedingly high and – in case of the studied B. subtilis model system – partly unknown level of cellular differentiation during bacterial biofilm development.