Peptidoglycan (PG), an essential glycopeptide polymer, is a fundamental component of the bacterial cell wall that maintains cell shape, provides structural integrity, and protects against osmotic lysis. PG biosynthesis is the target of several widely used antibiotics, and its degradation products are key modulators of host innate immune responses. This dissertation presents the development and application of advanced chemical and biological tools to study PG dynamics in both environmental and pathogenic bacterial systems. Building on prior work from the Grimes lab, PG labeling strategies were optimized using N-acetylmuramic acid (NAM)-based probes. Minimal tetrazine-functionalized NAM probes were utilized to broaden the labeling toolbox and enhanced labeling efficiency in Gram-positive bacteria using Bacillus subtilis as a model organism. Downstream workflows employing high-resolution microscopy, flow cytometry, and mass spectrometry were established to quantitatively and qualitatively assess PG labeling in isolated bacteria and in host-pathogen interaction models. To prove the role of PG recycling in bacterial pathogenesis, collaborations were established with Dr. Ramona Neunuebel and Dr. April Kloxin to investigate Legionella pneumophila strains expressing AmgK and MurU, enzymes critical for NAM salvage. This thesis work shows that AmgK is essential for intracellular replication of L. pneumophila in alveolar macrophage MH-S cells, underscoring its role in host adaptation. Furthermore, in collaboration with Prof. Ashu Sharma, AmgK from the oral pathogen Tannerella forsythia was investigated. Structural analysis revealed a unique ABSTRACT xxviii C-terminal domain, predicted to bind ATP, which was found to be essential for enzymatic function distinguishing it from canonical AmgK homologs. Collectively, these findings contribute to the understanding of bacterial cell wall dynamics, hostpathogen interactions, and offer new avenues for therapeutic development targeting diseases such as Legionnaires’ disease and periodontitis.