Co-Evolutionary Characterization of PBP2 as the Predominant Siderophore Recognizer in Diverse 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 analyzing siderophore uptake systems, first in five representative genera and then across a comprehensive dataset of 16,232 Gram-positive genomes. Through a pipeline integrating genome mining, coevolutionary analysis, and structural modeling, 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. Impact StatementBacteria secrete siderophores to scavenge iron and import the siderophore-iron complex via specific receptors, a process that shapes microbial community dynamics. However, predicting these interactions has been challenging, because the specific siderophore receptors in Gram-positive bacteria remained largely uncharacterized. In this study, we opened this "black box" by analyzing a comprehensive dataset of 16,232 genomes spanning the majority of Gram-positive bacteria. Through coevolutionary analysis, we identified PBP2 proteins as the primary "locks" that recognize siderophore "keys." We further demonstrate that these receptors exhibit greater evolutionary flexibility than their Gram-negative counterparts, frequently decoupled genomically from siderophore biosynthesis genes yet linked by transcriptional regulation. This discovery fills a critical knowledge gap, providing the missing link needed to map the global landscape of siderophore uptake potential and enable "sequence-to-ecology" prediction of iron-interaction networks in Gram-positive bacteria.