Elucidating biofilm pH dynamics in minimally buffered environments

Tran, Peter

doi:https://doi.org/10.21985/n2-x98r-x563

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Elucidating biofilm pH dynamics in minimally buffered environments

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Bacteria represent the most abundant form of life on Earth and have evolved to successfully colonize nearly every environmental niche. In doing so, bacteria predominately form multicellular communities known as biofilms, resulting in increased resilience, persistence, and emergent behaviors. Consequently, biofilms present an attractive target for engineering and synthetic biology, as understanding biofilm physiology can elucidate mechanisms enabling pathogenic biofilms and provide new modalities for multicellular control. While biofilms provide individual bacteria many advantages, the dense cellular proliferation can also create intrinsic metabolic challenges including excessive acidification. Because such pH stress is commonly masked in buffered laboratory media, it remains unclear how biofilms cope with minimally buffered natural environments. In this work, we develop several methods to interrogate biofilm physiology, metabolism, and underlying pH dynamics under minimally buffered conditions.This dissertation details methods for studying biofilm pH dynamics. In doing so, we establish a toolbox which enables observation of a previously unseen pH dynamic. The primary efforts towards that are 1) using metabolic flux analysis to study biofilm energy metabolism in situ and 2) employing a suite of genetic screens, -omics, and microscopy to elucidate biofilm pH dynamics. We report Bacillus subtilis biofilms overcome this intrinsic metabolic challenge through an active pH regulation mechanism. Specifically, we find that biofilms can modulate their extracellular pH to the preferred neutrophile range, even when starting from acidic and alkaline initial conditions, while planktonic cells cannot. We associate this behavior with dynamic interplay between acetate and acetoin biosynthesis and show that this mechanism is required to buffer against biofilm acidification. Furthermore, we find that buffering-deficient biofilms exhibit dysregulated biofilm development and increased sensitivity to antibiotics when grown in minimally buffered conditions. This dissertation elucidates an emergent pH regulation behavior in biofilms that enable them to develop and persist that in future work could be targeted to control and engineer biofilm growth.

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