Construction and Functional Characterization of a Heterologous Quorum Sensing Circuit in <i>Clostridium sporogenes</i>

Quorum sensing (QS) is a bacterial communication mechanism that regulates gene expression in a population density-dependent manner. Substantial progress has been made in the engineering of QS systems in Gram-negative bacteria, but development of engineered QS systems in Gram positive bacteria remains limited. In this study, the obligate anaerobic Gram-positive bacterium <i>Clostridium sporogenes</i> was engineered with the <i>Staphylococcus aureus</i> agr-QS system to enable density-dependent gene regulation. Using LC-MS/MS, we confirmed production of autoinducing peptides in the engineered <i>C. sporogenes</i> strain. A QS-regulated GFP reporter demonstrated activation of expression in response to both exogenous AIP addition and increasing cell density, confirming functional integration of the agr operon. A media refreshment experiment showed that replacing the culture supernatant delayed QS activation, highlighting the importance of signal accumulation. Moreover, we observed that a noncognate AIP from another agr specificity group acts as a competitive antagonist, inhibiting gene expression under the QS promoter. To our knowledge, this study presents the first successful engineering of the agr quorum sensing system in an obligate anaerobe, expanding the synthetic biology toolkit and offering new opportunities for bacterial therapies and metabolic engineering.