Jones, Alexander Joseph2015-08-242015-08-242015http://udspace.udel.edu/handle/19716/16984Clostridium acetobutylicum is a Gram-positive, anaerobic, spore-forming bacterium with potential in metabolic engineering. Recent work has been devoted to elucidating the regulatory and stress response mechanisms in C. acetobutylicum, with increasing focus on transcriptomics. 6S RNA and tmRNA are small, noncoding RNA molecules with observed influences on stress response in many prokaryotic organisms. 6S RNA regulates gene expression during the transition from exponential to stationary phase growth, and tmRNA contributes to quality control activities in cells under stress as a component of a ribosomal rescue system. In this study, we investigated the influence of 6S RNA (sCA_C1377) and tmRNA (sCA_C834) overexpression on tolerance to butanol in C. acetobutylicum ATCC 824. Strains overexpressing each sRNA sequence exhibited sustained higher percent survival under toxic concentrations of butanol stress through late stationary phase growth; this phenotype was particularly evident for 6S RNA overexpression. In addition to higher butanol tolerance, overexpression of 6S RNA also resulted in an increase in butanol production. Overexpression of a homologous 6S RNA sequence showed no discernible effects on tolerance or solvent production. In recent years, a sRNA sequence called solB (sCA_P176) has been under discussion as a negative regulator of solventogenesis genes in C. acetobutylicum. To investigate this putative regulatory function, we generated a solB overexpression strain and a solB deletion strain, the latter generated using a novel allelic exchange system in clostridia (Al-Hinai, et al, 2012). Overexpression of solB generated the anticipated solvent-negative phenotype, and quantitative PCR and Northern analysis revealed strong expression reduction of the solventogenic genes (adhE1, ctfA, and adc). Surprisingly, deletion of solB similarly produced no solvents, although without loss in solventogenic gene transcripts. Antisense knockdown of solB generated the same solvent-negative metabolic phenotype. Putative secondary structures and base-pairing predictions suggested respectable interaction between solB and the UTRs of each target transcript. Also, QPCR data revealed a possible early induction of solventogenic gene transcription in the deletion strain. Overall, this study indicates a clear role of solB on solventogenesis, but one that is more complex than initially thought; it is possible, given the results presented here, that a bimodal function for solB exists.Non-coding RNA.Clostridium acetobutylicum.Butanol.Solvents.Thesis919090644