Systematic characterization of PBP2 as the primary siderophore recognizer in Actinomycetes and other Gram-positive bacteria

Iron is a scarce yet essential nutrient for bacteria. Microbes often acquire iron by secreting siderophores, a diverse group of small molecules that form high-affinity complexes with iron for microbial uptake. Understanding microbial iron interaction networks requires detailed characterization of siderophore recognition specificity. In Gram-positive bacteria, substrate-binding proteins (SBPs) bind iron-siderophore complexes and deliver them to ABC transporters for import. However, the SBPs responsible for selective recognition remain poorly characterized, hindering large-scale data mining and network reconstruction. Here, we addressed this knowledge gap by systematically analysing siderophore uptake systems, first in five representative genera and then across a comprehensive dataset of 16,232 Gram-positive bacterial genomes. Through a pipeline integrating genome mining, coevolutionary analysis and structural modelling, we established PBP2 (Peripla_BP_2) subtype SBPs as the primary siderophore recognizer family. We revealed that, unlike the physically clustered systems in Gram-negative bacteria, synthetase and recognizer genes in Gram-positive bacteria are sometimes genomically decoupled, yet display coordinated transcriptional regulation by iron-responsive transcription factors. Our findings underscore key differences between Gram-positive and Gram-negative iron acquisition systems, providing foundational knowledge for large-scale inference of siderophore-mediated microbial interactions.