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.

Microbial competition exists in the general environment, such as soil or aquatic habitats, upon or within unicellular or multicellular eukaryotic life forms. The molecular actions that govern microbial competition, leading to niche establishment and microbial monopolization, remain undetermined. The emerging technology of imaging mass spectrometry (IMS) enabled the observation that there is directionality in the metabolic output of the organism Bacillus subtilis when co-cultured with Staphylococcus aureus. The directionally released antibiotic alters S. aureus virulence factor production and colonization. Therefore, IMS provides insight into the largely hidden nature of competitive microbial encounters and niche establishment, and provides a paradigm for future antibiotic discovery.

SpoIIIE is an FtsK-related protein that transports the forespore chromosome across the Bacillus subtilis sporulation septum. We use membrane photobleaching and protoplast assays to demonstrate that SpoIIIE is required for septal membrane fission in the presence of trapped DNA, and that DNA is transported across separate daughter cell membranes, suggesting that SpoIIIE forms a channel that partitions the daughter cell membranes. Our results reveal a close correlation between septal membrane fission and the assembly of a stable SpoIIIE translocation complex at the septal midpoint. Time-lapse epifluorescence, total internal reflection fluorescence (TIRF) microscopy, and live-cell photoactivation localization microscopy (PALM) demonstrate that the SpoIIIE transmembrane domain mediates dynamic localization to active division sites, whereas the assembly of a stable focus also requires the cytoplasmic domain. The transmembrane domain fails to completely separate the membrane, and it assembles unstable foci. TIRF microscopy and biophysical modeling of fluorescence recovery after photobleaching (FRAP) data suggest that this unstable protein transitions between disassembled and assembled oligomeric states. We propose a new model for the role of SpoIIIE assembly in septal membrane fission that has strong implications for how the chromosome terminus crosses the septum.[Keywords: SpoIIIE; DNA translocase; membrane fission; FRAP; PALM] Supplemental material is available at http://www.genesdev.org.

SpoIIIE is a membrane-anchored DNA translocase that localizes to the septal midpoint to mediate chromosome translocation and membrane fission during Bacillus subtilis sporulation. Here we use cell-specific protein degradation and quantitative photoactivated localization microscopy in strains with a thick sporulation septum to investigate the architecture and function of the SpoIIIE DNA translocation complex in vivo. We were able to visualize SpoIIIE complexes with approximately equal numbers of molecules in the mother cell and the forespore. Cell-specific protein degradation showed that only the mother cell complex is required to translocate DNA into the forespore, whereas degradation in either cell reverses membrane fission. Our data suggest that SpoIIIE assembles a coaxially paired channel for each chromosome arm comprised of one hexamer in each cell to maintain membrane fission during DNA translocation. We show that SpoIIIE can operate, in principle, as a bi-directional motor that exports DNA.DOI: http://dx.doi.org/10.7554/eLife.06474.001

Lambdoid phage 21 uses a pinholin-signal anchor release endolysin strategy to effect temporally regulated host lysis. In this strategy, the pinholin S 21 68 accumulates harmlessly in the bilayer until suddenly triggering to form lethal membrane lesions, consisting of S 21 68 heptamers with central pores <2 nm in diameter. The membrane depolarization caused by these pores activates the muralytic endolysin, R 21, leading immediately to peptidoglycan degradation. The lethal S 21 68 complexes have been designated as pinholes to distinguish from the micrometer-scale holes formed by canonical holins. Here, we used GFP fusions of WT and mutant forms of S 21 68 to show that the holin accumulates uniformly throughout the membrane until the time of triggering, when it suddenly redistributes into numerous small foci (rafts). Raft formation correlates with the depletion of the proton motive force, which is indicated by the potential-sensitive dye bis-(1,3-dibutylbarbituric acid)pentamethine oxonol. By contrast, GFP fusions of either antiholin variant irsS 21 68, which only forms inactive dimers, or nonlethal mutant S 21 68 S44C , which is blocked at an activated dimer stage of the pinhole formation pathway, were both blocked in a state of uniform distribution. In addition, fluorescence recovery after photobleaching revealed that, although the antiholin irsS 21 68-GFP fusion was highly mobile in the membrane (even when the proton motive force was depleted), more than one-half of the S 21 68-GFP molecules were immobile, and the rest were in mobile states with a much lower diffusion rate than the rate of irsS 21 68-GFP. These results suggest a model in which, after transiting into an oligomeric state, S 21 68 migrates into rafts with heterogeneous sizes, within which the final pinholes form.bacteriophage | membrane protein | membrane potential | structured illumination microscopy I n general, the phage infection cycle is terminated by the function of the holin, a small viral membrane protein (1). Throughout the morphogenesis period, holins accumulate harmlessly in the cytoplasmic membrane until suddenly forming lethal membrane lesions (holes). This event, called triggering, occurs at an allele-specific time. The lesions formed by canonical holins are very large, with diameters of micrometer-scale, allowing the escape of prefolded phage endolysins from the cytoplasm (2, 3). However, another important class of holins, the pinholins, form holes too small for the passage of protein. The results of a combined biochemical, genetic, ultrastructural, and computational approach indicate that the prototype pinholin, S 21 68 ( Fig. 1 A and B), of lambdoid phage 21 of Escherichia coli forms heptamers with a central lumen <2 nm in diameter (4), too small to allow the passage of endolysin (5). Phages using pinholins, therefore, require a distinct class of muralytic enzymes called the signal anchor release (SAR) endolysins, which are exported in a membrane-tethered enzymatically inactive form during the latent period. When the pinholins trig...