COS 28-1 - Towards understanding the ecological importance of metallophore-based zinc acquisition to support microbial community growth and ecosystem function

Abstract: Micronutrient metals such as iron and zinc are required by nearly all microorganisms, yet their bioavailability in many soil environments is low, provoking competition and limiting community growth and ecosystem functions. Microbial strategies for accessing iron have been well studied, and often involve the secretion of high-affinity iron-chelating compounds (siderophores) that solubilize iron and facilitate uptake. Since siderophores can only be utilized by the subset of microbes with dedicated membrane-bound transporters that recognize specific compounds, these molecules govern the competitive success and interactions of microbes in soils, thereby influencing microbial community structure. However, little is known about whether similar strategies are employed to access other metals, such as zinc. Here, we describe the chemical structure and biosynthetic pathway of a novel zinc-binding metallophore produced by a Pseudomonas sp. that facilitates zinc uptake in calcareous soil. We first detected the zinc-binding metallophore from nutrient enrichments using ultra-high-performance 21 Tesla Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). We then isolated and sequenced the producing bacteria, purified the zinc-binding metallophore, hereafter named ‘wardenbactin’, and elucidated its structure by tandem mass spectrometry and nuclear magnetic resonance analysis. Production of wardenbactin increased 45-fold under zinc-limited (5 nM Zn²⁺) growth conditions relative to zinc-replete (5,000 nM Zn²⁺) conditions, supporting slower but sustainable growth rates of the Pseudomonas sp. in the zinc-limited environment and suggesting a role in facilitating zinc uptake. Differential gene expression analysis from RNA-seq under zinc-limited growth conditions relative to zinc-replete conditions was used to identify an operon of 19 upregulated genes that encode proteins involved in the biosynthesis and transport of wardenbactin. Specifically, we propose that these proteins form a modular biosynthetic pathway for wardenbactin production based on its elucidated structure and the annotated biosynthesis operon. Ongoing work aims to generate knockout-gene mutants to validate the proposed pathway. While our results demonstrate that wardenbactin is involved in zinc acquisition in zinc-limited calcareous soil, this biosynthetic pathway is also conserved in numerous bacterial taxa found in terrestrial and marine ecosystems, thus underscoring the ecological importance of metallophore-based zinc acquisition to support community growth and ecosystem functions across many environments. Such molecules and biosynthetic genes may also be useful ecological markers of zinc deficiencies that indicate when certain ecosystem functions become limited by zinc availability to the microbial community.