Experimentele Evolution von Paracoccus denitrificans in anoxischen Chemostaten

Natural microbial communities play a central role in ecosystems and global cycles of elements. The microbial community compositions, functions as well as interactions between species and the environment have been studied with increasing effort. However, it is challenging to understand which parameters determine for the success of individual species to survive in a specific habitat. The often highly diverse microbial communities are continuously subjected to environmental stress such as biotic and abiotic fluctuations that cannot be completely tracked. To investigate the influence of different parameters on the ability of microorganisms to adapt to the environment, simple microbial communities, often single species are cultivated in the laboratory under strictly controlled conditions with reduced complexity. Such long-term experiments provide insight into the association between genetic and phenotypic alterations that evolve over hundreds or even thousands of generations. The availability of nutrients often affects microbial growth. This thesis describes the experimental evolution of Paracoccus denitrificans Pd1222, a model denitrifying soil bacterium, to study the adaptation on acetate or nitrate limitation. Initially, nutrient limitation for the anaerobic growth of P. denitrificans was addressed with focus on trace elements (Chapter 2). New trace element solutions were designed based on previous reports and tested to exclude growth limitation or inhibition by these nutrients during long-term cultivation. Improved generation times of 4.4 hours were achieved with a chelated trace element solution and lower concentrations than frequently used media. Chapter 3 describes the adaptive responses of P. denitrificans to acetate and nitrate limitation during experimental evolution in chemostats. In the course of at least 800 generations of P. denitrificans under denitrifying conditions the metabolic conversions of substrates were monitored. For deeper insights into different adaptive mechanisms of P. denitrificans under both conditions we investigated the transcriptomes and genome variations. Throughout the experiment the different treatments led to significantly different substrate conversion rates and transcriptomic profiles. Specifically, in nitrate limited cultures genes of the citric acid cycle and the nitrogen metabolism showed higher transcriptional activities than in acetate limited cultures. In the latter the transcription of genes encoding regulators and transporters was more pronounced. Additionally, more changes in transcriptional activities and in metabolism were observed over time than under nitrate limitation. Most notably, denitrification became more efficient resulting in the depletion of nitrite that accumulated in the culture during the first 500 generations. Although numerous mutations were detected in DNA obtained from this culture, they could not be related to the observed phenotypic changes. In all cultures the types and numbers of genetic variations did not considerably differ. The study indicated that P. denitrificans had a stronger potential to adapt to acetate limitation than to nitrate limitation and underlines the capacity of this bacterium to improve denitrification even in absence of environmental fluctuations. The possible explanation that phenotypic changes may have been independent of genetic variations is discussed in Chapter 4. The relevance of the insights gained in this study for natural, in particular denitrifying communities is presented and future studies towards the understanding of natural microbial community functions are suggested