Reconstitution of the S. aureus agr quorum sensing pathway reveals a direct role for the integral membrane protease MroQ in pheromone biosynthesis.

In virulence is under the control of a quorum sensing (QS) circuit encoded in the accessory gene regulator () genomic locus. Key to this pathogenic behavior is the production and signaling activity of a secreted pheromone, the autoinducing peptide (AIP), generated following the ribosomal synthesis and posttranslational modification of a precursor polypeptide, AgrD, through two discrete cleavage steps. The integral membrane protease AgrB is known to catalyze the first processing event, generating the AIP biosynthetic intermediate, AgrD (1-32) thiolactone. However, the identity of the second protease in this biosynthetic pathway, which removes an N-terminal leader sequence, has remained ambiguous. Here, we show that membrane protease regulator of QS (MroQ), an integral membrane protease recently implicated in the response, is directly involved in AIP production. Genetic complementation and biochemical experiments reveal that MroQ proteolytic activity is required for AIP biosynthesis in specificity group I and group II, but not group III. Notably, as part of this effort, the biosynthesis and AIP-sensing arms of the QS circuit were reconstituted together in vitro. Our experiments also reveal the molecular features guiding MroQ cleavage activity, a critical factor in defining specificity group identity. Collectively, our study adds to the molecular understanding of the response and virulence.