The bacterial cell is encased by an envelope that is in direct and constant contact with the environment. Thus, the envelope is the site where environmental fluctuations (e.g., changes in osmolarity) are first sensed by the bacterium. Also, components of the envelope mediate communication among other bacteria as well as commensal or pathogenic interactions between bacteria and their human hosts. These interactions play critical roles in complex environments such as the human gut microbiome. Finally, many of the most successful antibiotics in widespread use target the envelope (e.g., penicillin and vancomycin), and the efficacy of antibiotics with cytoplasmic targets is often reduced by issues with envelope permeability. For these reasons, the envelope is an area of intense research focus. Research approaches that investigate a single gene product or pathway have been immensely important in defining individual components of the envelope, but are limited in determining connections between pathways and are, by definition, low-throughput. Our lab (the Carol Gross lab at UCSF) previously took a systematic approach to investigate envelope function in the Gram-negative bacterium Escherichia coli by exposing a gene deletion library to a panel of environmental stressors that targeted the envelope. Using this dataset, we were able to make significant advances in our understanding of peptidoglycan (PG) synthesis, a key component of the bacterial cell wall. However, the envelope of Gram-positive bacteria, such as Bacillus subtilis, is fundamentally different from that of E. coli. The B. subtilis envelope lacs an outer membrane (and, thus, a distinct periplasmic space), includes teichoic acid polymers that are absent in E. coli, and contains a layer of PG that is several times thicker than found in E. coli. In this study, I will adapt the high-throughput approach previously used by our lab to investigate the B. subtilis envelope. First, I will systematically identify phenotypes for single deletion mutants of all non-essential genes in B. subtilis and a subset of double deletions. Then, I will use these datasets to investigate specific envelope processes such as proteolytic cascades that regulate ECF sigma factors, poorly characterized targets of the envelope-related two-component systems, functional redundancy amongst penicillin-binding proteins, and pathways involving the C55 undecaprenol phosphate carrier lipid. Finally, I will use a novel phenotype I discovered during construction of the B. subtilis deletion library to determine the function of ylaN, a gene involved in cell shape. These high-throughput methodologies will accelerate the assignment of phenotypes and functions to the large number of uncharacterized putative envelope genes in B. subtilis. The discoveries made in this study will likely extend to important Gram-positive human pathogens, as well as human commensals in the gut microbiome.