Abstract Cholera continues to be a global burden manifesting as acute diarrheal disease that impacts impoverished and destabilized regions. Bacteria including Vibrio cholerae utilize the Type II Secretion System (T2SS) for the secretion of a diverse array of effector proteins and toxins to adapt to environmental changes, notably cholera toxin the causative agent of cholera symptoms. While a variety of T2SS substrates including cholera toxin are fully secreted, a subset is retained on the cell surface. A recently identified group of diverse substrates found in some gram-negative bacteria including V. cholerae contains a homologous C-terminal domain, called the GlyGly- Cterm, that targets its passenger proteins to the cell surface. This newly identified domain is processed by rhombosortase, a subfamily of rhomboid protease, before T2SS transport. It remains unclear what sequence of the GlyGly-Cterm is required for surface localization, what, if any, enzymes in addition to rhombosortase are responsible for GlyGly-Cterm protein maturation, and importantly, why V. cholerae have this distinct surface- retention system. Model protein, VesB from V. cholerae, will be used to probe the molecular requirements of the GlyGly-Cterm domain by employing fluorescence microscopy, cell fractionation, and activity assays. Additional genes coding for putative enzymes possibly involved in processing of GlyGly-Cterm proteins have been identified in an ordered V. cholerae transposon library screen. This will be followed up on by generating clean gene deletions and assessing VesB surface localization. Quantitative and spatial proteomics as well as enzymatic assays will be used to characterize the role of these gene products. The importance of the GlyGly-Cterm will be probed by expressing GlyGly-Cterm proteins with and without this extension. Currently characterized cell-surface associated bacterial enzymes are almost exclusively retained by N-terminal lipidation, while the GlyGly-Cterm system represents a novel C-terminal cell-surface anchoring mechanism. The enzymes containing a GlyGly-Cterm are involved in a variety of functions contributing to V. cholerae environmental persistence and pathogenesis including nutrient acquisition, biofilm formation, and potentially maintenance of membrane integrity. The importance of GlyGly-Cterm proteins is demonstrated by disruption of rhombosortase, which results in impaired cell growth, reduced biofilm formation, and sensitivity to membrane stress. Characterization of this system has the potential to explain why some bacteria have evolved this unique cell-surface retention system and may provide insight into conserved mechanisms of C-terminal membrane anchoring in higher organisms.