Discovery of metallophore diversity in Microbulbifer in mixed culture with a coral pathogen using computational mass spectrometry and genome mining

Iron is an essential component of cellular biology. Thus, irons low bioavailability is a key evolutionary pressure guiding microbial dynamics in the marine environment. Among marine bacteria, Microbulbifer is an underexplored and functionally versatile bacterial genus, which is commonly associated with sponges, algae, corals and sediments. Previously, genome analyses have revealed that Microbulbifer spp. can degrade polymers and synthesize natural products. Despite their recognized potential to produce secondary metabolites, siderophores are yet to be identified in Microbulbifer, and their iron acquisition strategies remain largely unknown. Here, we developed a comprehensive mass spectrometry-based query language (MassQL) code to determine siderophore production by Microbulbifer spp. in mono- and mixed culture with a marine pathogen, which can be replicated for discovery of these compounds in any organism. Using this workflow, we discovered a new metallophore, which we named bulbichelin, as well as a suite of previously unreported petrobactins containing unprecedented longer chain length acylation on the central spermidine moiety. We applied genome mining methods to describe the biosynthesis of these compounds. Using metal infusion mass spectrometry, we show that bulbichelins bind a variety of metals. Notably, neither of these compounds were produced in a co-culture of Microbulbifer with coral-derived pathogen Vibrio coralliilyticus Cn52-H1. This observation suggests that Microbulbifer uses alternate strategies in a mixed community, such as siderophore piracy for metal acquisition. Understanding how siderophores shape interspecies interactions between Microbulbifer spp. and other marine organisms will aid in unraveling the chemical and catalytic versatility of this genus and adaptation in nutrient deplete marine environment.