There is a renewed interest in the study of Clostridium acetobutylicum due to its applicability in renewable and "greener" production methods for replacement fuels and industrial solvents. Furthermore, due to significant advances in C. acetobutylicum genetic technologies, it has ostensibly become the model clostridia for studying other solventogenic and pathogenic clostridia. Of considerable interest are the solventogenesis and sporulation differentiation programs, both of which are abolished in degenerate strains (lacking the 192-kb, 178-gene megaplasmid "pSOL1"). The operon and small genetic locus necessary for solvent formation has been identified and characterized, but the exact gene or operon necessary for sporulation remains a mystery. Knowledge of this gene or operon can be utilized for bioengineering a non-spore forming, solvent producing strain, ideal for industrial continuous fermentations.
Identifying this gene or operon necessitates the development of a functional pSOL1 library and a selection assay for screening spore forming cells. Our approach is to create a library of all pSOL1 genes/operons in degenerate strains M5 and DG1 (both lack pSOL1), and select for spore forming transformants. C. acetobutylicum is efficiently transformed by plasmids <10 kB in size, thus we restrict the library inserts to 5–6 kb in size. We claim a controlled approach because all fragments were generated via specific PCR primers that ensure complete pSOL1 coverage and uninterrupted representation of all genes and predicted operons under the control of their natural promoter. We developed two assays for screening spore forming cells: one based on chloroform chemical treatment and another by flow-cytometric analysis. We show that chloroform treatment disrupts any C. acetobutylicum cell that has not significantly advanced into sporulation such that they are unable to yield colony forming units when spread onto nutrient plates. Plasmid DNA is then isolated from colonies that survive the chloroform treatment and sequenced for the identification of the specific gene/operon. Although not yet widely used for prokaryotic analysis, we will show that high-throughput flow cytometry can be used for discerning single-cell morphology (and thus distinguish between vegetative and sporulating cells) based upon membrane potential, DNA content, membrane integrity, forward scatter and side scatter characteristics.