Characterization of Bacillus Subtilis Colony Biofilms Via Mass Spectrometry and Fluorescence Imaging

Colony biofilms of Bacillus subtilis are a widely used model for studying cellular differentiation. Here, we adapt matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) to examine the cellular and molecular heterogeneity in B. subtilis colony biofilms. From B. subtilis cells cultivated on a biofilm-promoting medium, we detected two cannibalistic factors, which were not found with the same strain in previous MALDI-MSI studies under different culturing conditions. Given the importance of cannibalism in matrix formation of B. subtilis biofilms, we employed a transcriptional reporter to monitor matrix-producing cell subpopulations using fluorescence imaging. These two complementary imaging approaches were applied to characterize three B. subtilis strains, including the wild type isolate NCIB3610 and two mutants with defective and enhanced biofilm phenotypes (Δspo0A and ΔabrB). Upon deletion of key transcriptional factors, correlated changes were observed in biofilm morphology, signaling and cannibalistic factor distribution, and matrix-related gene expression, providing new insights on cannibalism in biofilm development. This work underscores the advantages of using multimodal imaging to compare spatial patterns of both chemicals and protein expression patterns, obtaining information on cellular heterogeneity and function not available when single characterization methods are used.  We are adapting this method to high-throughput screening for synthetic biology and metabolic engineering applications.