(278c) Identifying the Genes Under the Control of the Master Sporulation Regulator Spo0A in Clostridium Acetobutylicum

The anaerobic, endospore-former Clostridium acetobutylicum has received renewed attention recently because of its long industrial history of producing the solvents acetone, butanol, and ethanol. Of particular interest is butanol, which could be used as a biofuel, because it has several characteristics that make it superior to ethanol as a transportation fuel. However, initiation of solvent production is also tied to the initiation of sporulation through the transcription factor Spo0A. Sporulation is an energy intensive process in which the cell undergoes asymmetric division to produce a dormant daughter cell, capable of surviving a variety of harsh environmental condition, within the mother cell. Upon completion of sporulation, the mother cell undergoes autolysis to release the mature spore. Because of the energy requirements and the death of the mother cell, sporulation is not a desirable trait from an industrial bioprocess point-of-view, and therefore solventogenesis needs to be decoupled from sporulation. When Spo0A is knocked out, both sporulation and solventogenesis are inactivated, but knocking out sporulation-specific targets downstream of Spo0A may decouple the two processes without harming solvent production. Currently though, most of the targets of Spo0A in C. acetobutylicum are not known. Here, we present the most detailed study of the regulon of Spo0A, all of the genes under the control of Spo0A, in C. acetobutylicum. To construct the regulon, we've combined chromatin immunoprecipitation (ChIP) with transcriptional profiling of knockout strains to generate a thorough list of genes controlled by Spo0A. Though the Spo0A binding motif is known, it is very common throughout the genome. By hybridizing enriched Spo0A-binding DNA, from the ChIP experiment, to whole genome microarrays, we've been able to determine which of these Spo0A motifs are actually targets of Spo0A. To filter out false positives, we performed the same ChIP experiment using the Spo0A antibody against the Spo0A knockout strain, SKO1. Finally, again using the whole genome microarrays, we compared the SKO1 strain against a σ^F knockout strain and a σ^E knockout strain, two downstream transcription factors under the control of Spo0A. By comparing the knockouts against each other, we were again able to limit false positives, by limiting the contamination of genes under the control of σ^F and σ^E in predicting the Spo0A regulon. Combining the data from these two experiments has given us the most complete list of genes under the control of Spo0A in C. acetobutylicum. We've also compared the Spo0A regulon from C. acetobutyicum against that from Bacillus subtilis, the model organism for endospore-forming bacteria, to highlight differences between the two organisms' sporulation programs.

Acknowledgment: Supported by NSF Grant CBET-0853490 "Differentiation Engineering for Beneficial Alteration of Cellular Traits"