(571ba) Development and Application of High-Throughput Flow-Cytometric Techniques for Analyzing Prokaryotic Cell Cultures

The improvement of microorganisms for specialty chemical production, bioremediation, and biofuel generation, remains an immediate scientific and industrial goal. Particularly for bacteria, the goal of converting low value biomass into transportation fuels (ethanol and butanol) has motivated a tremendous amount of bacteria strain development. Subsequently, forward genetics and genome scale approaches are gaining considerable popularity for accelerating the discovery phase of strain development. A popular approach is to generate mutant, plasmid, or species libraries, and then screen for desirable phenotypes via environmental stress enrichment. Although often successful, these approaches are flawed by single parameter detection (fitness), and are typically not high-throughput. Flow cytometry epitomizes high-throughput and is ideal for multi-parametric detection of individual cells. Unfortunately, flow cytometric analysis of prokaryotic organisms is not as developed as for eukaryotes.

Thus, we developed and adapted multiple flow cytometry assays for the analysis of bacterial cell cultures. To accomplish this task, we employed the bacterium, Clostridium acetobutylicum. C. acetobutylicum exhibits a well known cycle of cellular differentiation consisting of at least four unique morphologies. Our goal was to develop an assay capable of identifying all four morphologies. Through light scattering and a modified BacLight? (Invitrogen) DNA staining, we definitively accomplish this task and also demonstrate the efficacy of an alternative approach via pencillin-binding-protein (PBP) detection. In general, we demonstrate the high-throughput and multi-parametric capabilities of these assays. Additionally, we demonstrate their application in fluorescence assisted cell sorting, generating enriched populations for electron microscopy analysis, and analyzing cultures in a manner previously not possible. In future work we hope to utilize these and other assays in screening and sorting mutant libraries for desirable phenotypes, for which environmental stress alone could not enrich.