Award: OCE-1722761

Intellectual Merit: Iron is an essential micronutrient for phytoplankton that is required for photosynthesis and respiration. Insufficient iron has been shown to limit phytoplankton growth in large regions of the surface ocean, and correspondingly, iron cycling is directly linked to carbon cycling in much of the marine environment. Nearly all iron in seawater (>99%) exists as complexes with organic molecules called ligands, which govern the concentration of iron dissolved in the water and the bioavailability of that iron to phytoplankton. However, despite the importance of iron-binding organic ligands, their sources and identities are largely unknown. Viruses, the majority of which are phages (viruses that infect bacteria), are extremely abundant in seawater and are in the same size fraction as dissolved iron. Recent evidence that non-marine phages contain iron as part of their structures led us to propose that marine phages may represent a previously overlooked class of organic iron-binding ligands. This model, dubbed the Ferrojan Horse Hypothesis, suggests that marine phages significantly contribute to dissolved iron pools and hypothesizes that phages may utilize bacterial iron-uptake receptors for infection in the manner of a Trojan horse. Finally, if a portion of the cellular iron thought to be released from bacterial cells for remineralization following lysis is already incorporated into phage particles, then these findings will have significant implications for oceanic biogeochemical models. This project successfully developed culture-based methods for tracing iron from bacteria into phage using the model system Escherichia coli and its lytic phages T4 and T5. Phage T4 has been shown in prior structural biology studies to contain iron in its tail fibers. While there is no structural evidence for iron in phage T5, this phage is known to use a siderophore-bound iron uptake receptor for infection. E. coli were grown exclusively on media containing labeled iron (Fe-57) for multiple generations such that the bacterial cells were highly enriched in labeled iron. In iron-free media, the bacteria were then infected by either phage T4 or phage T5 and the newly produced phage particles were purified using filtration, ultracentrifugation, and dialysis. The Fe-57 content in the final, purified phage particles was determined by Inductively coupled plasma mass spectrometry (ICP-MS) and the number of resulting phage particles was determined by SYBR Gold staining and epifluorescence microscopy to determine (1) how much iron is incorporated into phage particles and (2) if this iron originates from the bacterial host cell during infection. The presence of labelled iron in both T4 and T5 phage particles demonstrates its origin from within the bacterial host cell, with each of these different phage types incorporating hundreds to thousands of iron atoms per phage particle. The use of this model phage-host system shows that phage likely represent significant iron-binding ligands, although the strength of this interaction still needs further investigation. In addition, these studies demonstrate that the amount of bioavailable iron released from a bacterial cell during lysis is less than previously thought, since a portion of that iron will be incorporated into progeny phage particles. To bridge the gap between the E. coli model systems and our target study system of the oceans, we worked with Vibrio natriegens, a common marine/estuarine bacterium and emerging model system due to its rapid growth under laboratory conditions. We have optimized V. natriegens growth and obtained four lytic phages for use in further experiments, including newly isolating and sequencing the genome of a phage from Tampa Bay, Florida. Broader Impacts: As the first study to examine the biogeochemical impact of trace elements contained within the structure of highly abundant phage particles, this research has major implications for biological and chemical oceanography. These results also have far-reaching implications for other disciplines, including human health where iron availability plays an important role in microbial pathogenesis. This project contributed to the multidisciplinary training of a graduate student, an undergraduate student, two postdoctoral researchers, and a technician. Two peer-reviewed articles and a Master’s thesis resulted from this funding, and research results were disseminated in over ten scientific presentations. A public outreach activity about phage-host interactions was created and run during hands-on exhibits for the annual St. Petersburg Science Festival and several local youth groups. Last Modified: 03/25/2020 Submitted by: Mya Breitbart