and cell lysis are also linked to the copynumber of the plasmid containing the colicin operon. Theoretical and experimental analysis identified the global regulatory protein CsrA as main player, specifically the abundance of free CsrA determines lysis gene translation and thus, the delay between toxin production and release present in the ColicinE2 system. Even though CsrA is a highly abundant protein within the bacte-rial cell [78], only a small number of free CsrA is present [74]. Therefore, even small changes in the extent of CsrA sequestering elements can have a big impact on the number of free CsrA molecules that are able to repress cel gene translation. Further-more, the data presented in this thesis shows that the lysis time of colicin producing strains is coupled to the metabolic state of the cells due to changing CsrA abundances.
Changing the level of observation from single-cell behavior to single-strain population dynamics showed that cea and cel switching is dependent on the post-transcriptional regulation. For early cel switching, a high amount of cells lyse which results in differ-ent effective growth rates of colicin producing strains. This constitutes an important parameter for competition. In addition, a positive linear correlation between lysis time and toxin amount being released were found, which means that for later cell lysis, a higher amount of toxin is released into the environment.
In a next step, it was investigated how the single-cell TED affect competition of the toxin producing strain with a toxin sensitive strain. The impact of delayed lysis time and thus higher toxin amounts on two-strain competition between a toxin-sensitive S strain and three different CXstrains was studied for two growth media (varying carbon source). Long-term competition experiments in combination with theoretical analysis were performed similar to von Bronk et al. [10, 11]. No significant change between competition outcomes of the three strains was observed. However, the theoretical anal-ysis used in this study revealed that the two parameters lanal-ysis time and released toxin amount coupled in experiments were not equally important. In contrast, parameter sweeps revealed that the main factor for competition and lysis delay is the accumu-lation of a sufficient toxin amount. Even though simuaccumu-lation and experimental results showed good accordance without stress and at high stress, at intermediate induction levels larger discrepancies between theory and experiments were detected. Mainly in the model C wins fractions were underestimated. In earlier studies by Mader et al.
2015 it was shown that at medium stresses the ColicinE2 system expression is highly heterogeneous [13]. Therefore, when the model was adjusted for increased variability of the switching rate dCon, increasing C wins fractions were found at intermediate induction levels. Even though the adjusted model does not entirely describe the
ob-served experimental results, this showed that heterogeneity in toxin expression and release dynamics on the single-cell level can have a big impact on competition out-come, increasing the success of colicin producers.
Based on the fact that variability can be an important feature increasing competi-tive success of the C strain, the noise of the ColicinE2 operon was investigated. The coefficient of variation of the FIs of a reporter strain SREP1 with regulation similar to the wild-type ColicinE2 operon and various mutants were compared to identify regulation mechanisms controlling noise generation within the system. Three types of noise were defined according to switching behavior as noise of the OFF state, ON state and population noise. For the strain closest to the wild-type colicin system OFF state noise of cea is bigger than for cel. For all other noise types, the noise of cel exceeds that of cea, showing that when cells switch into the toxin producing state, noise of toxin production is smaller than noise of toxin release. Analyzing the response of strains carrying different mutations in the transcriptional and post-transcriptional regulation modules showed that similar to the expression dynamics, CsrA is a main player regulating not only noise of cel gene expression but also acts as global factor reducing noise of the entire colicin operon including cea and cel noise. Comparing data of different mutant strains and plasmid copynumbers showed that the natural system does have a significant noise level, indicating the presence of an optimal noise level that helps to increase the success of the C strain in bacterial competition.
In conclusion, the main regulation factor of both TED and noise generation in the ColicinE2 system was found to be the global regulation protein CsrA. Availability of free CsrA controls cell lysis and with it the amount of toxin that is released by colicin producers. This abundance is coordinated by a multitude of CsrA sequestering ele-ments and also connected to the metabolic state of a cell. Competition experiele-ments for strains with different plasmid composition and for two media showed comparable competition results for a broad range of induction levels and lysis times. This could indicate that changes in TED with its complex regulation help to increase C strain suc-cess for varying conditions. Theoretical analysis showed that as long as enough toxin is produced C strain can succeed for both comparable growth of toxin producers and toxin-sensitive strains but also if the C strain has a disadvantage in competition due to a smaller growth rate. Expression and release of the colicin were shown to be highly noisy and the theoretical model showed that increased variability in release times at intermediate stresses can give the colicin producers an advantage in competition. As the ColicinE2 systems main regulators were determined to be global, chromosomally
encoded proteins like LexA and CsrA, the observed characteristics might be important regulation mechanisms for a multitude of genes like other colicins or many plasmid encoded systems. In summary, this thesis showed that many regulatory factors work together to tune singe-cell TED that shape not only population fate of one strain, but can lead to long term competition success of the toxin producing population in varying environments.