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Firmicutes are gram-positive bacteria with important roles in human health, disease, and industry. However, more than a quarter of genes in the model firmicute Bacillus subtilis remain completely uncharacterized, including numerous core phylum-specific genes. Here, we design a compact pooled CRISPRi library targeting all protein-coding genes in B. subtilis and test its growth in ~150 stress conditions. Using data from this screen as a hypothesis generator, we perform targeted experiments that reveal that YneF, a conserved essential firmicute protein, plays a role in the SRP co-translational protein secretion pathway. We also demonstrate that ECF-transporters play a previously unknown but broadly conserved role in cell wall homeostasis, perform an unbiased analysis of amino acid crossfeeding, and make additional discoveries about bacterial competition and about the cell wall of B. subtilis. In addition to these major contributions to our understanding of B. subtilis (and gram-positive firmicutes in general), this work provides a rich dataset that will nucleate future studies of uncharacterized genes and presents a framework for accessible full-genome functional genomic screens in other bacteria. ### Competing Interest Statement The authors have declared no competing interest. National Institutes of Health, https://ror.org/01cwqze88, R35 GM118061 The University of Queensland, https://ror.org/00rqy9422