| dc.description.abstract | Complex I is a big protein complex in the electron transport chain and works as the entry point of electrons from NADH into the electron transport chain. Complex I in Cereibacter sphaeroides differs from complex I in many other bacteria, as there are two gene clusters that code for the protein complex: complex IA and complex IE. Both isozymes consist of the subunits NuoA – NuoN.
C. sphaeroides was randomly mutagenized using transposon mutagenesis during a teaching class at Ohio State University, and two of the mutants obtained were Sp10-28-8 E2 and SP10-JAI. These are, however, believed to be the same mutant, with the transposon insertion at nuoN in the gene cluster coding for complex IE. C. sphaeroides was also randomly mutagenized in the Alber laboratory. A mutant isolated from this screen was called ABC21CM32 and had the transposon insertion at a gene with an unknown function in the gene cluster coding for complex IA. This mutant was phenotyped to not grow aerobically with D-lactate as the carbon source.
The aim of this experiment was to phenotype Sp10-28-8 E2, Sp10-JAI, and ABC21CM32 by performing growth experiments with different carbon sources. To further investigate the function of complex I, a clean in-frame deletion of nuoE in the gene cluster coding for complex IE was performed. A third approach to study the carbon assimilation of C. sphaeroides was to perform a transposon mutagenesis experiment and map the transposon insertion sites on mutants which did not grow on certain carbon substrates.
The results of the growth experiments were surprising, as the phenotype of ABC21CM32 was different from the phenotype that was found last year. The phenotype of Sp10-28-8 E2/Sp10-JAI was also different from what was expected. Based on the experiment performed in this thesis, the preculture of the growth experiment influences the results.
Single crossovers of nuoE were successfully obtained, but due to lack of time, a double crossover was not obtained. Three transposon mutants that showed compromised growth on acetate were isolated, and the transposon insertion sites were mapped to be on genes which have an unknown function in C. sphaeroides’ ability to use acetate as a carbon source. | |
