Biofilm Formation by Staphylococcus aureus is Triggered by a Drop in the Levels of the Second Messenger cyclic-di-AMP

The bacterial pathogen Staphylococcus aureus forms multicellular communities known as biofilms in which cells are held together by an extracellular matrix. The matrix consists of repurposed cytoplasmic proteins and extracellular DNA. These communities assemble during growth on medium containing glucose, but the intracellular signal for biofilm formation was unknown. Here we present evidence that biofilm formation is triggered by a drop in the levels of the second messenger cyclic-di-AMP. Previous work identified genes needed for the release of extracellular DNA, including genes for the cyclic-di-AMP phosphodiesterase GdpP, the transcriptional regulator XdrA, and the purine salvage enzyme Apt. Using a cyclic-di-AMP riboswitch biosensor and mass spectrometry, we show that the levels of the second messenger drop during biofilm formation in a glucose-dependent manner and that the drop is prevented in mutants of all three genes. Importantly, we also show that expression of the "accessory gene regulator" operon agr is under the positive control of cyclic-di-AMP and that an agr mutation, which is known to promote biofilm formation, bypasses the block in biofilm formation and eDNA release caused by a gdpP mutation. We conclude that the effect of the glucose-dependent drop in c-di-AMP levels is principally mediated by a reduction in agr expression, which in turn promotes biofilm formation.