Abstract Biofilms are surface-bound microbial communities that are typically embedded in a matrix of self-produced extracellular polymeric substances and can cause chronic infections. Extracellular DNA is known to play a crucial role in biofilm development in diverse bacteria; however, the existence and function of RNA are poorly understood. Here, we show that RNA contributes to the structural integrity of biofilms formed by the human pathogen Staphylococcus aureus. RNase A dispersed both fresh and mature biofilms, indicating the importance of RNA at various stages. RNA-sequencing analysis demonstrated that the primary source of RNA in the biofilm matrix was the Brain Heart Infusion medium (>99.32%). RNA purified from the medium promoted biofilm formation. Microscopic and molecular interaction analyses demonstrated that polysaccharides were critical for capturing and stabilizing external RNA in biofilms, which contributes to biofilm organization. These findings provide a basis for exploring the role of externally derived substances in bacterial biofilm organization.
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