The transition from unicellular to multicellular life is captivating because free-living individuals become complex, coordinated assemblages that display unique properties and behaviors. It is a transformative step in biology that optimizes survival and resource utilization, especially in fluctuating environments. In microbiology, this multicellular organization assumes an intriguing form known as biofilms. Bacterial biofilms, assemblages of cells encased in a self-produced matrix, are sophisticated structures that provide protection from environmental challenges. The emerging understanding of biofilms reveals that bacteria within them do not exist as passive, isolated entities. Instead, they display spatial organization, physiological differentiation, and even metabolic interactions such as cross-feeding. The pathogenic bacterium Pseudomonas aeruginosa, which is a common cause of biofilm-based infections and a popular model organism, has been shown to form metabolic subpopulations and differentially regulate gene expression across depth in biofilms. However, one open question is the nature of this cellular arrangement in P. aeruginosa biofilms, the mechanisms governing it, and its physiological ramifications. My thesis addresses the overarching question: Does cellular arrangement in P. aeruginosa biofilms influence nutrient distribution, metabolic activity, antibiotic tolerance, and metabolic cross feeding? Through the use of paraffin embedding, thin-sectioning, and confocal microscopy, I delve deep into the biofilm, particularly in the z-direction, byproducing high-resolution images that provide insights into the three-dimensional structure and dynamics of these bacterial communities. The first chapter, serving as the foundation of this exploration, provides an introduction of the principles of multicellularity. It draws attention to the hallmarks of multicellularity, encompassing metabolic cross-feeding, protective advantages, and labor specialization while also shedding light on its challenges. In the context of multicellularity, biofilms are introduced, emphasizing the formation of bacterial biofilms, their environmental and medical implications, and specifically highlighting the importance of P. aeruginosa biofilms for understanding microanatomy and physiology. Chapter 2 presents the crux of our exploration, underlining how cellular arrangement directly impacts metabolic activity and antibiotic tolerance in P. aeruginosa biofilms. A striking observation was the presence of vertical, clonal striations, suggesting the presence of an organized architecture within mature biofilms. Mutants with disordered cell arrangements, particularly in O-antigen attachment, showed altered patterns of nutrient distribution and metabolic activity in addition to distinct patterns of antibiotic- induced cell death. Such findings build on prior knowledge by illuminating the intricate relationships between biofilm anatomy, metabolic differentiation, and drug tolerance. Chapter 3 introduces the use of light-sheet microscopy for live imaging of pellicle biofilms, which offers a real-time window into biofilm development and cellular dynamics. In Chapter 4, the narrative takes a broader perspective, focusing on the influence of various carbon sources on cellular arrangement. It introduces the presence of metabolic cross-feeding among different biofilm subpopulations and hints at the potential relationship between cell arrangement and heterogeneous metabolic activity patterns. The work in this thesis reveals that the arrangement of cells within P. aeruginosa biofilms determines metabolic outcomes, antibiotic responses, and potential cross- feeding interactions. In a world where biofilm-related infections account for an alarming 80% of persistent bacterial infections, understanding biofilm microanatomy has implications for therapeutic strategies and possibly reshaping our battle against antibiotic tolerance. A more detailed picture of the relationship between cell arrangement, physiological differentiation, and metabolic cooperation within biofilms has the potential to provide inroads toward new approaches to combating these recalcitrant structures.

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