2007-05-092007-05-091995Wong, John. "Genetic regulation in Clostridium acetobutylicum ATCC 824." (1995) Diss., Rice University. <a href="https://hdl.handle.net/1911/16899">https://hdl.handle.net/1911/16899</a>.https://hdl.handle.net/1911/16899Genetic Regulation in Clostridium acetobutylicum was studied to understand what signals caused this bacterium to switch between different growth stages. A biochemical approach was employed to study DNA supercoiling, while a molecular approach was employed to study sigma factors. Cells treated with novobiocin, a gyrase inhibitor, produced higher butyrate levels and lower butanol and acetone levels. Seven enzyme activities involved in acid and solvent production were tested; CoA transferase, which catalyzes acetone formation and acid uptake, experienced a 50% decrease in activity. However, Northern analysis showed that CoA transferase mRNA levels did not decrease to a higher extent than mRNA levels of other enzymes involved in acid and solvent production, suggesting that the regulation was at the post-transcriptional level. The data also suggest that acetone and alcohol production may be regulated by different mechanisms. Three genes were cloned from C. acetobutylicum into Escherichia coli and sequenced. Two of them encoded proteins that show high homology with two Bacillus subtilis sporulation-specific sigma factors, $\rm\sigma\sp{E}$ and $\rm\sigma\sp{G}$ and the third one encoded a putative $\sigma\sp{E}$-processing enzyme. The gene arrangement of these three genes was conserved in two Bacillus species. Duplication and inactivation of these genes were then attempted. While the wild-type strain eventually ceased to grow and produce solvents in batch cultures (in test tubes) over time, the mutant strains showed sustained growth for a much longer time and produced a threefold increase in acetone and butanol. By regulating sporulation, $\rm\sigma\sp{E}$ and additional sigma factors may play a role in both solvent induction and termination. A model was proposed suggesting an overlap between signals causing cells to produce solvents and to sporulate. Loss of such signals may lead to degenerate cells. By understanding more about the genetic regulation of this organism, it is possible to perform metabolic engineering to increase its potential to be used in biotechnology.application/pdfengCopyright is held by the author, unless otherwise indicated. Permission to reuse, publish, or reproduce the work beyond the bounds of fair use or other exemptions to copyright law must be obtained from the copyright holder.MicrobiologyThesisTHESIS BIOCHEM. 1995 WONG