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bioRxiv · 10.64898/2026.05.13.724699

The phenotypic landscape of the model firmicute Bacillus subtilis

Kim, L. M.·Koo, B.-M.·Burkhart, H. N.·Vollmer, D.·Vollmer, W.·Todor, H.·Gross, C. A.
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Abstract

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, revealing that the conserved essential firmicute protein YneF is part of 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. In addition to these major contributions to our understanding of B. subtilis biology (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. SIGNIFICANCELarge-scale chemical genomics screens facilitate the characterization of genes by generating phenotypic data across a library of gene mutants. Here, using CRISPRi, we designed a compact pooled library targeting all genes in B. subtilis and screened growth of the library in [~]150 conditions. Importantly, we used this dataset to expand B. subtilis biology on levels ranging from molecular pathways to bacterial communities. We discovered a role for an essential firmicute gene in protein secretion, implicated two new players in cell wall homeostasis, and unraveled fundamental factors driving competition and cooperation in the soil. This rich dataset will serve as a hypothesis generator, expanding our set of bacterial phenotypic data and driving future experiments in B. subtilis and other gram-positive firmicutes.

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Kim, L. M., Koo, B.-M., Burkhart, H. N., Vollmer, D., Vollmer, W., Todor, H., Gross, C. A.. 2026-05-14. The phenotypic landscape of the model firmicute Bacillus subtilis. https://doi.org/10.64898/2026.05.13.724699

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