Fatty acid synthesis knockdown promotes biofilm wrinkling and inhibits sporulation in Bacillus subtilis

Many bacterial species typically live in complex three-dimensional biofilms, yet little is known about systematic changes to gene function between non-biofilm and biofilm lifestyles. Here, we created a CRISPRi library of knockdown strains covering all known essential genes in the biofilm-forming Bacillus subtilis strain 3610. We show that gene essentiality is largely conserved between liquid and surface growth and between two media. We developed an image analysis algorithm to quantify biofilm colony wrinkling, which identified strains with high or low levels of wrinkling that were uncorrelated with extracellular matrix gene expression. We also designed a high-throughput screen for sensitive quantification of sporulation efficiency and performed the first screens of sporulation during essential gene knockdown. We found that all basal knockdowns of essential genes were competent for sporulation in a sporulation-inducing medium, but certain strains exhibited reduced sporulation efficiency in LB, a medium with generally lower levels of sporulation. Knockdown of fatty acid synthesis increased wrinkling and inhibited sporulation. These results highlight the importance of essential genes in biofilm structure and sporulation/germination and suggest a previously unappreciated and multifaceted role for fatty acid synthesis in bacterial lifestyles and developmental processes. Abstract ImportanceFor many bacteria, life typically involves growth in dense, three-dimensional communities called biofilms that contain cells with differentiated roles and are held together by extracellular matrix. To examine how gene function varies between non-biofilm and biofilm growth, we created a comprehensive library of strains using CRISPRi to knockdown expression of each essential gene in the model species Bacillus subtilis 3610, which can develop into a wrinkled biofilm structure or a spore capable of surviving harsh environments. This library enabled us to determine when gene essentiality depends on growth conditions. We also developed high-throughput assays and computational algorithms to identify essential genes involved in biofilm wrinkling and sporulation. Knockdown of fatty acid synthesis increased the density of wrinkles, and also inhibited sporulation in a medium with generally lower sporulation levels. These findings indicate that essential processes such as fatty acid synthesis can play important and multifaceted roles in bacterial development.