The phenotypic landscape of the model firmicute Bacillus subtilis.

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 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, revealing that the conserved essential firmicute protein YneF is part of the SRP cotranslational protein secretion pathway. We also demonstrate that ECF-transporters play a broadly conserved role in cell wall homeostasis, perform an unbiased analysis of amino acid crossfeeding, and make additional insights into bacterial competition. This work additionally provides a rich dataset to nucleate future studies of uncharacterized genes and presents a framework for performing accessible full-genome functional genomic screens in other bacteria.