| Bacteria, like humans, can exhibit 'individuality' when faced with similar choices. The presence of positive and negative feedback in regulatory systems plays a crucial role in dictating 'individuality' in bacteria. Interestingly, positive and negative feedback loops are also present in sugar utilization pathways ubiquitous to virtually all microorganisms. The bacterial sugar utilization pathways encode regulators, transporters and catabolic enzymes that are induced by their cognate sugars. The regulators control the expression of the pathway components; transporters import more sugar conferring positive feedback and the enzymes break down the sugar imparting negative feedback. Because of scientific and biotechnological implications, sugar utilization pathways in the model bacterium Escherichia coli have been studied for many decades. However, most of the work done so far involved bulk characterization techniques that fail to capture unique behaviors observable only at the single-cell level. The very few single-cell studies available were focused on only two pathways. More importantly, the studies neglected sugar catabolism, which is an integral part of the natural sugar utilization pathways. Therefore, it remains unclear how the myriad of natural sugar utilization pathways respond to sugars at the single-cell level. This thesis work addresses this gap by investigating the single-cell response of E. coli to eight different sugars using a combination of experimental and computational approaches. The response of E. coli to sugars was remarkably diverse, with negative feedback due to catabolism playing a crucial role. Building on these insights, we engineered cells to convert sharp bimodal responses of natural sugar utilization pathways into linear, titratable responses. The motivation behind the work was to use the natural inducible sugar utilization pathways as 'in-built' titratable systems in non-model organisms. Using the L-arabinose and D-xylose utilization pathways as model systems, we showed that each modification came with a trade-off. Finally, as an extension to this work we showed how the inadvertent presence of inducer in the medium could affect the response properties such as linearity and dynamic range. Overall, this work provides a few significant insights (a) feedback loops in bacterial sugar utilization are one of the crucial factors that help the cells attain 'individuality' or cope under fluctuating nutrient conditions (b) contrary to the prevailing assumptions, response of these ubiquitous utilization pathways can be extremely complex, with potential effects on how the cells can be used for various biotechnological applications (c) the endogenous sugar utilization pathways, with modifications, can be co-opted as titratable systems especially in non-model microorganisms. As part of future work, the strength of the feedbacks loops could be tuned to understand their effects on the transition rates between induced and uninduced states. Also, the single-cell analyses could be extended to microorganisms beyond E. coli to verify the generality of the observed diverse responses. |
