A comprehensive mass spectrometry-based query language (MassQL) code is developed to determine siderophore production by Microbulbifer spp.
Abstract
Iron is an essential component of cellular biology. Thus, iron's low bioavailability is a key evolutionary pressure guiding microbial dynamics in the marine environment. Among marine bacteria, Microbulbifer is a chemically 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 cultures. Using this workflow, we discovered a new metallophore, which we named bulbichelin, as well as a suite of previously unreported petrobactins containing an 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. 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.
Trace metals are essential for microbial physiology, yet their limited availability drives the evolution of metallophores, which are specialized molecules for metal acquisition. This thesis explores siderophore structure and function using and advanced LC-MS experimental workflows. First, escherichelin, a metabolite from Escherichia coli Nissle 1917, has been shown to possess zinc-binding potential. Second, marine Microbulbifer species were found to encode conserved RiPP biosynthetic clusters that produce bulbicupramide, a thiooxazole-containing peptide that selectively binds Cu(I), suggesting a chalkophore-like role in benthic ecosystems. Finally, magnetite nanoparticles (Fe₃O₄) were synthesized and optimized for siderophore enrichment under alkaline conditions, demonstrating strong selectivity for desferrioxamine. Untargeted metabolomics confirmed enrichment of diverse secondary metabolites, highlighting the utility of nanoparticle-based platforms for selective metabolite class isolation.
Microbial natural products are the source of over 70% of all known antibiotics, yet the pace of their discovery has slowed significantly since its peak in the mid-20th century. This stagnation is largely due to the repeated isolation of known compounds from readily culturable microorganisms, while the vast majority of microbial biosynthetic gene clusters (BGCs) remain silent and unexpressed under typical laboratory conditions. The convergence of genomics, synthetic biology, and high resolution analytical chemistry now provides a powerful toolkit to unlock this cryptic biosynthetic potential. This thesis presents a strategy that integrates these disciplines to awaken silent BGCs and discover novel bioactive molecules.
A genome mining approach was utilised to identify 22 promising BGCs from diverse actinobacteria, prioritised for their predicted novelty. To activate their expression, a suite of synthetic biology and molecular cloning strategies was implemented in both native and engineered heterologous hosts. This systematic activation campaign yielded several significant outcomes: (i) the linking of three known compounds to their previously unknown BGCs; and (ii) the discovery and structural elucidation of two novel natural products. Notably, one of the compounds represents a new class of calcium-dependent antibiotics with potent antimicrobial activity.
In conclusion, this research demonstrates the efficacy of a genome-led approach to drug discovery. It has successfully translated genomic data into tangible chemical matter, functionally characterised previously cryptic BGCs, and contributed a novel class of antibiotics to the global pipeline for combating infectious diseases.
Marine sponges are known sources of bioactive natural products (NPs), many of which are produced by associated bacterial symbionts via encoded biosynthetic gene clusters (BGCs). A particularly interesting subclass of sponge-derived NPs is comprised of small, brominated alkaloids, which are recovered from diverse habitats and host sponge taxonomies. Despite having been described decades ago, most of these NPs do not have an elucidated biosynthetic origin. We queried metagenomes of several sponge species by making use of a minimal set of core enzymes that we postulate to be necessary to produce these small peptidic NPs: an FADH2-dependent halogenase and an AMP-binding adenylation enzyme. This revealed a variety of novel BGC architectures, many of which showed conservation among sponge host phylogenies and were encoded in the genomes of diverse sponge-associated bacteria. Furthermore, we identified a BGC in the sponge G. barretti that is potentially linked to the production of the iconic barettins, given its enzymatic machinery and specific acidobacterial origin. The present work contributes to the challenging quest to link orphan brominated NPs to their parent BGCs in the sponge holobiont and beyond.
Catarina Loureiro, M. Schorn, Mohammad M. Alanjary et al.· bioRxiv· 0 citations
The integrated bioinformatics pipeline enabled the reconstruction of 37 medium-to-high-quality metagenome-assembled genomes (MAGs), and recovered 147 BGCs mostly from Pseudomonadota, Actinomycetota, and Acidobacteriota phyla, highlighting the Siwa Oasis as a promising reservoir of unexplored biosynthetic potential and a valuable resource for natural product discovery to address global health challenges.
Muhammad A. Ajagbe, Shimaa F Ahmed, Amged A. Ouf et al.· World Journal of Microbiolog...· 0 citations
Bacteria employ specialized metabolites called siderophores to acquire scarce iron, but these molecules may serve additional ecological roles. Here, we reveal that Pandoraea species, including environmental isolates and opportunistic pathogens typically acquired from the environment, produce bifunctional lipopeptides that undergo enzymatic remodeling to switch from promoting bacterial motility to optimizing iron capture. Through genome mining and metabolic profiling, we discovered pandorachelins, diazeniumdiolate-containing siderophores. Comprehensive NMR analysis, derivatization, and isotope labeling established that pandorachelin A is a head-to-tail-fused homodetic cyclopeptide, revising a recently proposed structure. We identified the elusive biosynthetic precursor, pandorachelin B, as a lipocyclopeptide with a lactone moiety and N-terminal fatty acid. A specialized acylase (PdnM) cleaves the lipid tail of pandorachelin B, triggering an O→N acyl shift that contracts the ring and transforms the biological function: the lipopeptide enables bacterial swarming through surfactant activity, while the delipidated product exhibits enhanced iron-chelating capacity but no motility promotion. Genetic knockouts, enzyme reconstitution, and phenotypic assays confirm this maturation sequence. The functional switch correlates with ecological niche across Pandoraea species, revealing a sophisticated strategy for niche colonization and nutrient acquisition. These findings identify PdnM as a potential antivirulence target and expand the functional repertoire of bacterial siderophores.
Elena Herzog, K. Ishida, Evelyn M. Molloy et al.· Angewandte Chemie· 0 citations