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Secondary metabolites are important facilitators of plant–microbe interactions in the rhizosphere, contributing to communication, competition, and nutrient acquisition. However, at first glance, the rhizosphere seems full of metabolites with overlapping functions, and we have a limited understanding of basic principles governing metabolite use. Increasing access to the essential nutrient iron is one important, but seemingly redundant role performed by both plant and microbial Redox-Active Metabolites (RAMs). We used coumarins, RAMs made by the model plant Arabidopsis thaliana, and phenazines, RAMs made by soil-dwelling pseudomonads, to ask whether plant and microbial RAMs might each have distinct functions under different environmental conditions. We show that variations in oxygen and pH lead to predictable differences in the capacity of coumarins vs phenazines to increase the growth of iron-limited pseudomonads and that these effects depend on whether pseudomonads are grown on glucose, succinate, or pyruvate: carbon sources commonly found in root exudates. Our results are explained by the chemical reactivities of these metabolites and the redox state of phenazines as altered by microbial metabolism. This work shows that variations in the chemical microenvironment can profoundly affect secondary metabolite function and suggests plants may tune the utility of microbial secondary metabolites by altering the carbon released in root exudates. Together, these findings suggest that RAM diversity may be less overwhelming when viewed through a chemical ecological lens: Distinct molecules can be expected to be more or less important to certain ecosystem functions, such as iron acquisition, depending on the local chemical microenvironments in which they reside.

Microbes that live on plant roots can make important contributions to plant health and often exist in tight-knit communities held together by chemical exchanges. This study investigates an interaction between two such metabolites: the climate-active gas nitrous oxide (N 2 O) and cobalamin. N 2 O can become toxic through a reaction with methionine synthase enzymes that use cobalamin as a cofactor. We asked whether the production of N 2 O by some bacteria curtails the growth of others that rely on these enzymes. Using genetic mutants of a model bacterium and natural isolates from the roots of the plant Arabidopsis thaliana, we showed that N 2 O-producing microbes suppress growth of their sensitive neighbors and that N 2 O sensitivity is common in rhizosphere bacteria. As natural and agricultural soils periodically experience bursts of N 2 O, our results suggest that exposure to this gas may shape the assembly of plant-beneficial microbial communities.

Vitamin B 1 (thiamin) is a cofactor for critical enzymatic processes and is scarce in surface oceans. Several eukaryotic marine algal species thought to rely on exogenous thiamin are now known to grow equally well on the precursor 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP), including the haptophyte Emiliania huxleyi . Because the thiamin biosynthetic capacities of the diverse and ecologically important haptophyte lineage are otherwise unknown, we investigated the pathway in transcriptomes and two genomes from 30 species representing six taxonomic orders. HMP synthase is missing in data from all studied taxa, but the pathway is otherwise complete, with some enzymatic variations. Experiments on axenic species from three orders demonstrated that equivalent growth rates were supported by 1 µM HMP or thiamin amendment. Cellular thiamin quotas were quantified in the oceanic phytoplankter E. huxleyi using the thiochrome assay. E. huxleyi exhibited luxury storage in standard algal medium [(1.16 ± 0.18) × 10 −6  pmol thiamin cell −1 ], whereas quotas in cultures grown under more environmentally relevant thiamin and HMP supplies [(2.22 ± 0.07) × 10 −7 or (1.58 ± 0.14) × 10 −7  pmol thiamin cell −1 , respectively] were significantly lower than luxury values and prior estimates. HMP and its salvage-related analog 4-amino-5-aminomethyl-2-methylpyrimidine (AmMP) supported higher growth than thiamin under environmentally relevant supply levels. These compounds also sustained growth of the stramenopile alga Pelagomonas calceolata . Together with identification of a salvage protein subfamily (TENA_E) in multiple phytoplankton, the results indicate that salvaged AmMP and exogenously acquired HMP are used by several groups for thiamin production. Our studies highlight the potential importance of thiamin pathway intermediates and their analogs in shaping phytoplankton community structure. IMPORTANCE The concept that vitamin B 1 (thiamin) availability in seawater controls the productivity and structure of eukaryotic phytoplankton communities has been discussed for half a century. We examined B 1 biosynthesis and salvage pathways in diverse phytoplankton species. These comparative genomic analyses as well as experiments show that phytoplankton thought to require exogenous B 1 not only utilize intermediate compounds to meet this need but also exhibit stronger growth on these compounds than on thiamin. Furthermore, oceanic phytoplankton have lower cellular thiamin quotas than previously reported, and salvage of intermediate compounds is likely a key mechanism for meeting B 1 requirements under environmentally relevant scenarios. Thus, several lines of evidence now suggest that availability of specific precursor molecules could be more important in structuring phytoplankton communities than the vitamin itself. This understanding of preferential compound utilization and thiamin quotas will improve biogeochemical model parameterization and highlights interaction networks among ocean microbes. The concept that vitamin B 1 (thiamin) availability in seawater controls the productivity and structure of eukaryotic phytoplankton communities has been discussed for half a century. We examined B 1 biosynthesis and salvage pathways in diverse phytoplankton species. These comparative genomic analyses as well as experiments show that phytoplankton thought to require exogenous B 1 not only utilize intermediate compounds to meet this need but also exhibit stronger growth on these compounds than on thiamin. Furthermore, oceanic phytoplankton have lower cellular thiamin quotas than previously reported, and salvage of intermediate compounds is likely a key mechanism for meeting B 1 requirements under environmentally relevant scenarios. Thus, several lines of evidence now suggest that availability of specific precursor molecules could be more important in structuring phytoplankton communities than the vitamin itself. This understanding of preferential compound utilization and thiamin quotas will improve biogeochemical model parameterization and highlights interaction networks among ocean microbes.

The secretion of small Fe-binding molecules called siderophores is an important microbial strategy for survival in Fe-limited environments. Siderophore production is often regulated by quorum sensing (QS), a microbial counting technique that allows organisms to alter gene expression based on cell density. However, the identity and quantities of siderophores produced under QS regulation are rarely studied in the context of their roles in Fe uptake. We investigated the link between QS, siderophores, and Fe uptake in the model marine organism Vibrio harveyi where QS is thought to repress siderophore production. We find that V. harveyi uses a single QS- and Fe-repressed gene cluster to produce both cell-associated siderophores (amphiphilic enterobactins) as well as several related soluble siderophores, which we identify and quantify using liquid chromatography-coupled (LC)-MS as well as tandem high-resolution MS (LC-HR-MS/MS). Measurements of siderophore production show that soluble siderophores are present at ∼100× higher concentrations than amphi-enterobactin and that over the course of growth V. harveyi decreases amphienterobactin concentrations but accumulates soluble siderophores. 55Fe radio-tracer uptake experiments demonstrate that these soluble siderophores play a significant role in Fe uptake and that the QS-dictated concentrations of soluble siderophores in stationary phase are near the limit of cellular uptake capacities. We propose that cell-associated and soluble siderophores are beneficial to V. harveyi in different environmental and growth contexts and that QS allows V. harveyi to exploit “knowledge” of its population size to avoid unnecessary siderophore production.

It is common for bacteria to produce chemically diverse sets of small Fe-binding molecules called siderophores. Studies of siderophore bioinorganic chemistry have firmly established the role of these molecules in Fe uptake and provided great insight into Fe complexation. However, we still do not fully understand why microbes make so many siderophores. In many cases, the release of small structural variants or siderophore fragments has been ignored, or considered as an inefficiency of siderophore biosynthesis. Yet, in natural settings, microbes live in complex consortia and it has become increasingly clear that the secondary metabolite repertoires of microbes reflect this dynamic environment. Multiple siderophore production may, therefore, provide a window into microbial life in the wild. This minireview focuses on three biochemical routes by which multiple siderophores can be released by the same organism—multiple biosynthetic gene clusters, fragment release, and precursor-directed biosynthesis—and highlights emergent themes related to each. We also emphasize the plurality of reasons for multiple siderophore production, which include enhanced iron uptake via synergistic siderophore use, microbial warfare and cooperation, and non-classical functions such as the use of siderophores to take up metals other than Fe.

Cryptogamic species and their associated cyanobacteria have attracted the attention of bio-geochemists because of their critical roles in the nitrogen cycle through symbiotic and asymbiotic biological fixation of nitrogen (BNF). BNF is mediated by the nitrogenase enzyme, which, in its most common form, requires molybdenum at its active site. Molybdenum has been reported as a limiting nutrient for BNF in many ecosystems, including tropical and temperate forests. Recent studies have suggested that alternative nitrogenases, which use vanadium or iron in place of molybdenum at their active site, might play a more prominent role in natural ecosystems than previously recognized. Here, we studied the occurrence of vanadium, the role of molybdenum availability on vanadium acquisition and the contribution of alternative nitrogenases to BNF in the ubiquitous cyanolichen Peltigera aphthosa s.l. We confirmed the use of the alternative vanadium-based nitrogenase in the Nostoc cyanobiont of these lichens and its substantial contribution to BNF in this organism. We also showed that the acquisition of vanadium is strongly regulated by the abundance of molybdenum. These findings show that alternative nitrogenase can no longer be neglected in natural ecosystems, particularly in molybdenum-limited habitats.

Microbes frequently navigate the environment with the help of small, excreted metabolites. Iron-binding molecules called siderophores are one such set of secondary metabolites that are commonly used by microbes to access the essential trace element iron. Although many marine microbes produce siderophores, a substantial number, including the highly abundant SAR11 clade of Pelagibacterales, do not and it has remained unclear whether such nonproducers can access siderophore-bound iron. Here, we show that iron-limited SAR11 cultures fail to grow in the presence of the hydroxamate siderophore ferrichrome but exhibit robust growth in the presence of the catechol siderophore enterobactin. We confirm that this is linked to iron availability using transcriptomic and 55Fe radio tracer uptake experiments. This phenotype can be explained by the relative lability of enterobactin-bound iron in seawater, a phenomenon that has been previously observed in field studies and which we demonstrate with a simple kinetic model. Further experiments with the marine heterotrophs Phaeobacter inhibens and Vibrio harveyi suggest that enterobactin-Fe is unlikely to support the faster growth rates of these organisms without the use of biochemical uptake mechanisms. Overall, our work provides a model of siderophore use that considers bioavailability conferred through both kinetic and biochemical mechanisms and shows that some catechol-bound Fe may be widely available to small, slow growing marine organisms.

Biological nitrogen fixation, the main natural input of fixed nitrogen into the biosphere, is catalyzed by Mo-, V-, or Fe-only nitrogenase metalloenzymes. Although “alternative” V- and Fe-only nitrogenase genes are found in many environments, the contribution of these isoenzymes to N₂ fixation is unknown. Here we present a new method (ISARA, isotopic acetylene reduction assay) that distinguishes canonical Mo and alternative nitrogenase activities based on in vivo ¹³C fractionation of acetylene reduction to ethylene (¹³εMₒ = 13.1–14.7 ‰, ¹³εV = 7.5–8.8 ‰, ¹³εFₑ = 5.8–6.5 ‰). ISARA analyses indicate significant contributions of alternative nitrogen fixation in boreal cyanolichens and salt marshes (~10–40 % acetylene reduction, ~20–55 % N₂ fixed). These results affect the quantitative interpretation of natural abundance ¹⁵N data or traditional acetylene reduction assays. They also invite a reexamination of the conditions under which the different nitrogenase isozymes are active and suggest significant interactions between the cycles of nitrogen and trace metals.

A variety of trace metals, including prominently iron (Fe) are necessary for marine microorganisms. Chemically defined medium recipes have been used for several decades to study phytoplankton, but similar methods have not been adopted as widely in studies of marine heterotrophic bacteria. Medium recipes for these organisms frequently include tryptone, casamino acids, as well as yeast and animal extracts. These components introduce unknown concentrations of trace elements and organic compounds, complicating metal speciation. Minimal medium recipes utilizing known carbon and nitrogen sources do exist but often have high background trace metal concentrations. Here we present H-Aquil, a version of the phytoplankton medium Aquil adapted for marine heterotrophic bacteria. This medium consists of artificial seawater supplemented with a carbon source, phosphate, amino acids, and vitamins. As in Aquil, trace metals are controlled using the synthetic chelator EDTA. We also address concerns of EDTA toxicity, showing that concentrations up to 100 µM EDTA do not lead to growth defects in the copiotrophic bacterium Vibrio harveyi or the oligotrophic bacterium Candidatus Pelagibacter ubique HTCC1062, a member of the SAR11 clade. H-Aquil is used successfully to culture species of Vibrio, Phaeobacter, and Silicibacter, as well as several environmental isolates. We report a substantial decrease in growth rate between cultures grown with or without added Fe, making the medium suitable for conducting Fe-limitation studies in a variety of marine heterotrophic bacteria.

The need for living things to obtain trace elements creates a fundamental interaction between Life and Earth. Iron (Fe), and a handful of other metals, are used ubiquitously in biochemistry, yet must be extracted from insoluble minerals. The major biogeochemical cycles occurring at the Earth’s surface are also catalyzed by metalloenzymes. Trace elements therefore form one of the strongest links in the coupling of geologic and biologic processes and their use is key to understanding the co-evolution of Life and Earth. This thesis explores two microbial solutions to the problem of trace metal scarcity: the substitution of different trace elements in enzymes and the production of siderophores or small molecules that aid in trace metal uptake. I use high-throughput sequencing and newly developed isotopic techniques to determine that ‘alternative’ nitrogenases – containing vanadium (V) or Fe-only instead of molybdenum (Mo) – can make substantial (>20%) contributions to nitrogen fixation and nitrogenase diversity in coastal sediments, raising questions about their overall role in nitrogen cycling. My experiments with nitrogen-fixing Azotobacter vinelandii cultures show that the siderophore protochelin is co-regulated by limitation for both Fe and the nitrogenase cofactor Mo. Protochelin complexes Mo, and up-regulation under Mo-limitation is consistent with its long hypothesized role as a molybdophore. Additionally, I report that A. vinelandii can invest > 30% of fixed nitrogen in siderophores and that this nitrogen is isotopically distinct from biomass. Under conditions of iron-limitation siderophore production changes the isotopic composition (δ15N) of A. vinelandii biomass, a result that may help to explain variations in δ15 N from laboratory and field studies of diazotrophs. Finally, I investigate the regulation of siderophore production by Fe and quorum sensing (QS, a microbial counting technique that allows bacteria to tailor their gene expression to their cell density). I find that the marine bacterium Vibrio harveyi uses a single gene cluster to produce both strong, cell-bound siderophores as well as weak soluble siderophores and that QS allows V. harveyi to calibrate its siderophore production to its cellular iron uptake capacity. This final chapter highlights ‘biotic’ and ‘abiotic’ controls on siderophore production and the potential importance of microbial interactions in geobiological processes.