Horizontal gene transfer is a key mechanism in bacterial genome evolution, enabling adaptation through gene exchange between cells. Among them, bacterial conjugation significantly contributes to the dissemination of antibiotic resistance. Conjugation requires direct cell-to-cell contact mediated by a type IV secretion system (T4SS) encoded by the integrative and conjugative element (ICE), which assembles the secretion apparatus and transfers DNA. We use ICESt3 from Streptococcus thermophilus as a model to dissect conjugation. Here, we focus on OrfA, a peptidoglycan hydrolase, to elucidate its function in this process. Sequence analysis revealed two predicted catalytic domains in OrfA: a lysozyme-like domain and a CHAP (cysteine–histidine–dependent amidohydrolase/peptidase) domain. Mutants with individual disruptions in each domain were generated in S. thermophilus to assess their impact on conjugation. Both mutants exhibited undetectable conjugation frequencies, and complementation restored transfer to wild-type levels. This demonstrates that both Lyt and CHAP domains are required for efficient DNA transfer and supports a role for both in the precise remodeling/cleavage of peptidoglycan necessary for proper T4SS functionality. To better understand how the CHAP domain functions, we performed a lytic activity assay and observed inhibition by specific metal ions. To characterize potential CHAP–cation interactions, we employed thermal shift assays (TSA) and switchSENSE. TSA with multiple ions revealed decreases in thermal stability consistent with specific protein–ion interactions, and switchSENSE (static mode) supported direct binding to the OrfA CHAP domain. Ongoing dynamic-mode switchSENSE and near-UV circular dichroism will assess whether these interactions are accompanied by conformational changes.