Biofilms are structured microbial communities embedded in a self-produced extracellular matrix, whose architecture emerges from the interaction between genetic regulation, collective behavior, and physical constraints. This lifestyle enables bacteria to persist across various environments, including industrial and medical settings, and confined systems such as space habitats. Aboard the International Space Station, scientists discovered biofilm formation in life-support systems, highlighting the importance of understanding how physical forces, such as altered gravity, affect biofilm formation and function.

In this study, we investigated the genetic determinants of biofilm fitness under altered gravity using a CRISPR interference (CRISPRi) approach in Bacillus subtilis NDmed. Biofilm fitness was quantified as relative abundance in pooled CRISPRi screens during biofilm growth. Building on prior transcriptomic analyses, we designed a CRISPRi library targeting more than 400 coding and non-coding genetic elements that are upregulated during the early stages of biofilm formation. We first validated our system, designated CUTE, as an effective tool for controlling gene expression and testing biofilm-associated genes in different biofilm models. This analysis provided insight into the role of uncharacterized non-coding RNA elements in biofilm development and highlighted the importance of genes involved in matrix synthesis, motility, chemotaxis (including aerotaxis), cell wall synthesis, genetic regulation, and the stress response in solid-to-air macrocolony and liquid-to-air pellicle models in NDmed.

We then performed pooled CRISPRi screens to identify key genes under Earth gravity, simulated micro- and hyper-gravity. Different biofilm models were subjected to various gravity conditions approximating those of the ISS (near 0 g), Mars (0.38 g), the Moon (0.16 g), and a range of increasing gravity conditions from 2 g to 16 g. Gravity levels were simulated using a Random Positioning Machine and a Large-Diameter Centrifuge at the European Space Agency. Altered gravity reshaped gene fitness, with strong dependence on biofilm model and structure. Sessile communities, including macrocolonies, surface-attached biofilms, and pellicles, exhibited architecture-dependent fitness responses consistently involving genes linked to metabolism, regulation, and matrix-associated functions, albeit with distinct contributions across models. In contrast, populations associated with swarming motility showed a distinct pattern, with stronger contributions from transcriptional and translational processes. Our screens did not reveal any universally gravity-responsive genes across the tested models, suggesting that gravity may indirectly influence biofilm formation by modulating physical boundary conditions and altering the physical and physiological context of the microbial community.

Overall, this study establishes pooled CRISPRi screening as a functional genomics framework for studying biofilm formation under altered gravity and shows that an architecture-aware perspective, in which gene fitness emerges from interactions between spatial organization and physical constraints, is critical for interpreting microbial responses in space-relevant environments and future life-support infrastructures. Finally, this study illustrates how spaceflight-driven research can provide broadly applicable principles for understanding and controlling biofilms in terrestrial medical, industrial, and environmental systems.