Bacteria frequently encounter changes in their environment and must adapt accordingly. When these changes are predictable, there is evidence of anticipatory gene regulation. For example, the model enteric pathogen Salmonella has a well-defined natural history, typically only encountering iron in the lumen of the gut. However, this bacterium responds to the presence of iron not by upregulating genes needed to thrive in the lumen, but rather the iron-deplete epithelium, the subsequent environment it encounters. This may be similar to Pavlovian conditioning, a type of associative learning that involves pairing two unrelated stimuli and anticipatory behavioural changes. Since conditioning has not been well explored in bacteria, we are investigating whether Salmonella can learn new conditioned responses by pairing two unrelated carbon sources, citrate and maltose. We leveraged a prototype of the Microbial Evolution and Growth Arena (MEGA)-plate motility assay to define a new natural history. By pairing stimuli across a physical landscape, we can select for bacteria that learn to use citrate to anticipate maltose and can quickly deplete the second carbon source. Time-series imaging of bacteria as they swim across the plate shows evidence of emerging variants capable of swimming faster through maltose and unique swimming behaviours through repeated passaging. This approach selects for the fastest swimming bacteria, not necessarily bacteria that have acquired anticipatory regulation. As such, further genetic and transcriptional analysis of the variants are necessary. Similar passaging of Salmonella in broth allowed us to compare anticipatory regulation across a physical and non-physical landscape. Learning to anticipate environmental changes will provide a bacterium with a selective advantage, allowing it to outcompete its conspecifics which are slower to respond. From this investigation, we hope to provide insight into the learning capacity of bacteria and further understand how bacteria exploit memory to problem-solve. / Thesis / Master of Science (MSc) / Bacteria that encounter predictable changes in their environment can acquire anticipatory gene regulation. This may be similar to Pavlovian conditioning, a type of associative learning that involves pairing two unrelated stimuli and anticipatory behavioural changes. Since conditioning has not been well explored in bacteria, we investigate whether Salmonella can learn new behaviours by pairing two unrelated carbon sources, citrate and maltose. We leveraged a motility plate assay to define a new natural history. By pairing stimuli across a physical landscape, we can select for bacteria that learn to use citrate to anticipate maltose. Time-series imaging shows evidence of variants capable of swimming faster through maltose and unique swimming behaviours through repeated passaging. Similar passaging of Salmonella in broth allowed us to compare anticipatory regulation across physical and non-physical landscapes. From this investigation, we hope to further understand the learning capacity of bacteria and how bacteria exploit memory to solve problems.