Single-cell approach dissecting agr quorum sensing dynamics in Staphylococcus aureus

This ENA submission contains MiSeq whole genome sequences of eight Staphlococcus aureus strains. NM-I, NM-II, NM-III and NM-IV are congenic strains in the Newman (ST8) background. The native agr locus was deleted and replaced with the agr-system of any of the four types. The strains originate from the study: https://doi.org/10.1128/jb.06685-11 The other four are agr prototype strains (LAC: agr-I, ST8; 502a: agr II, ST5; MW2: agr III, ST1; MNTG: agr IV, ST2276) containing the agr-P3-YFP reporter plasmid used in this study. Abstract: Quorum sensing (QS) enables bacteria to coordinate collective behaviors by secreting and sensing diffusible signals. Understanding QS at single-cell resolution is essential because population-level measurements often obscure regulatory heterogeneity. In Staphylococcus aureus, the accessory gene regulator (agr) system is a major QS-controlled virulence regulator activated by autoinducing peptides (AIPs). Four agr-types exist, each defined by distinct AIPs and capable of cross-inhibition, yet their activation dynamics and interaction hierarchies remain poorly understood. Using microfluidics, time-lapse microscopy, and deep-learning-based image analysis, we quantified agr-activation in congenic and native agr-type strains. Agr-types differed in sensitivity to their homologous AIPs: agr-III was largely unresponsive, whereas agr-IV was highly sensitive with elevated basal activation. Agr-activation distribution was frequently bimodal (simultaneous existence of agr-ON and agr¬ OFF cells), driven by subpopulations that never activated or switched to agr-OFF despite constant stimulation. Combining homologous and heterologous AIPs, AIP-IV suppressed pre-activated agr-I, while AIP-I had no inhibitory effect on agr-IV, indicating asymmetric cross-inhibition. In spatially segregated cocultures, diffusional crosstalk resulted in four reproducible interaction regimes, stable-dominance, stable-, delayed-, or unstable-dual-activation, determined by agr-type pairing and influenced by genetic background. Our approach links single-cell signaling to population outcomes and uncovers agr-type-specific asymmetries with potential consequences for strain competition and virulence.