Primer on agar-based microbial imaging mass spectrometry.

Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) imaging mass spectrometry (IMS) applied directly to microbes on agar-based medium captures global information about microbial molecules, allowing for direct correlation of chemotypes to phenotypes. This tool was developed to investigate metabolic exchange factors of intraspecies, interspecies, and polymicrobial interactions. Based on our experience of the thousands of images we have generated in the laboratory, we present five steps of microbial IMS: culturing, matrix application, dehydration of the sample, data acquisition, and data analysis/interpretation. We also address the common challenges encountered during sample preparation, matrix selection and application, and sample adherence to the MALDI target plate. With the practical guidelines described herein, microbial IMS use can be extended to bio-based agricultural, biofuel, diagnostic, and therapeutic discovery applications.

Application of bacterial cytological profiling to crude natural product extracts reveals the antibacterial arsenal of Bacillus subtilis.

Although most clinically used antibiotics are derived from natural products, identifying new antibacterial molecules from natural product extracts is difficult due to the complexity of these extracts and the limited tools to correlate biological activity with specific molecules. Here, we show that bacterial cytological profiling (BCP) provides a rapid method for mechanism of action determination on plates and in complex natural product extracts and for activity-guided purification. We prepared an extract from Bacillus subtilis 3610 that killed the Escherichia coli lptD mutant and used BCP to observe two types of bioactivities in the unfractionated extract: inhibition of translation and permeablization of the cytoplasmic membrane. We used BCP to guide purification of the molecules responsible for each activity, identifying the translation inhibitors bacillaene and bacillaene B (glycosylated bacillaene) and demonstrating that two molecules contribute to cell permeabilitization, the bacteriocin subtilosin and the cyclic peptide sporulation killing factor. Our results suggest that bacillaene mediates translational arrest, and show that bacillaene B has a minimum inhibitory concentration 10 × higher than unmodified bacillaene. Finally, we show that BCP can be used to screen strains on an agar plate without the need for extract preparation, greatly saving time and improving throughput. Thus, BCP simplifies the isolation of novel natural products, by identifying strains, crude extracts and fractions with interesting bioactivities even when multiple activities are present, allowing investigators to focus labor-intensive steps on those with desired activities.