Abstract
<jats:p> <jats:italic>Staphylococcus aureus</jats:italic> biofilm formation is a key factor enabling persistent infections. However, the lack of efficient high-throughput tools previously limited systematic study of its regulatory mechanisms. Here, we used high-efficiency transduction to construct two luminescent bioreporter libraries, each probing a distinct biofilm regulatory pathway. Derived from the Nebraska Transposon Mutant Library, these libraries enabled rapid, quantitative screening of biofilm-associated gene expression in a high-throughput format. Our screens revealed a surprising lack of overlap in the regulation of the two biofilm components investigated: adhesin synthesis and extracellular DNA production. However, we identified <jats:italic>mntR</jats:italic> as a key gene involved in the expression of both biofilm components and confirmed the previously reported role of <jats:italic>yjbH</jats:italic> . Cross-lineage validation showed that these regulators retain conserved significance across multiple <jats:italic>S. aureus</jats:italic> backgrounds, although their phenotypic effects varied across strains. Collectively, this work provides a versatile, high-throughput framework to dissect the regulatory networks underlying complex phenotypes in <jats:italic>S. aureus</jats:italic> . </jats:p>