Carbon Catabolism in <i>Bacillus subtilis</i>: Global and Molecular Views on the Control of Gene Expressiondissertation
Аннотация: The soil bacterium B. subtilis is exposed to frequently changing environmental conditions. Moreover, in its natural habitat, B. subtilis is surrounded by numerous microorganisms that are competing for the available resources. Therefore, the survival strategy of B. subtilis is optimized towards a rapid utilization of the available nutrients. The uptake of glucose, which is the preferred carbon source of B. subtilis, is mediated by the phosphoenolpyruvate:phosphotransferase system (PTS) encoded by the ptsGHI operon. The expression of this operon is controlled by transcriptional antitermination. The mRNA of the ptsGHI operon can adopt one of two alternative secondary structures in its 5' untranslated region. The formation of a thermodynamic more stable terminator causes premature transcription termination. However, when the antiterminator protein GlcT binds to this region, it stabilizes an antiterminator structure called RAT and thus prevents terminator formation. The activity of GlcT is controlled by the availability of glucose. Only in the presence of glucose, GlcT can bind and stabilize the RAT structure. There are three more PTS-dependent antitermination systems in B. subtilis. These systems are controlled by the availability of sucrose or β-glucosides. Both the antiterminator proteins as well as the respective RATs are all very similar in structure and sequence. Nevertheless, the regulation by the antiterminator proteins is highly specific.The aim of this work was the detection of all specificity determinants of the related antitermination systems. Site directed mutagenesis of two different RATs should help to identify nucleotides that are essential for the specific interaction with the respective antiterminator proteins. Furthermore, it was tested if the specificity of certain RATs can be changed towards other antiterminator proteins. For the simultaneous introduction of multiple point mutations, a method called MMR (multiple mutation reaction) was established. All nucleotides essential for specificity are located in the lower loop regions of the related RAT structures. Site directed mutagenesis of this region could also change specificity of certain RATs towards other antiterminator proteins. The interaction of the antiterminator proteins with their respective permeases is also specific and contributes to the overall specificity of the systems. Furthermore, it was found that carbon catabolite repression is also essential for the maintenance of specificity and for preventing cross-talk among the different systems. For the utilization of its preferred carbon source glucose, B. subtilis features a complete set of enzymes for glycolysis, the pentose phosphate shunt, the tricarboxylic acid cycle and the respiratory chain. When available, glucose causes the repression of genes needed for the utilization of alternative carbon sources. Only when glucose is completely exhausted, the genes necessary for the utilization of these carbon sources are expressed. This effect is known as carbon catabolite repression and has already been intensively studied.The second aim of this work was to determine the effect of the organic acids glutamate and succinate on the central metabolism of B. subtilis when given in addition to glucose. This was achieved by a combined approach using transcriptomic data and metabolic flux analysis. The results of both studies were in good agreement for most of the studied genes. The addition of glutamate and succinate had no major effect on the genes of glycolysis and the pentose phosphate pathway. However, the flux of acetyl-CoA into the tricarboxylic acid cycle was severely reduced. On the other hand, the overflow metabolic pathways of lactate and acetate synthesis were significantly induced. For some of the genes, the change in transcription had not the expected consequence on the metabolic fluxes. This is in particular true for the genes involved in acetoin biosynthesis. Although the relevant genes were highly induced, no formation of acetoin was observed. These differences in the two data sets could indicate that the relevant enzymes are regulated on the level of protein activity.
Год издания: 2007
Авторы: Oliver Schilling
Ключевые слова: Bacterial Genetics and Biotechnology, Bacteriophages and microbial interactions, Enzyme Structure and Function
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