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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.

Most biological nitrogen transformations have characteristic kinetic isotope effects used to track these processes in modern and past environments. The isotopic fractionation associated with nitrogen fixation, the only biological source of fixed nitrogen (N), provides a particularly important constraint for studies of nitrogen cycling. Nitrogen fixation using the ‘canonical’ Mo-nitrogenase produces biomass with a δ^(15)N value of ca. −1‰ (vs. atmospheric N_2). If the ‘alternative’ V- and Fe-only nitrogenases are used, biomass δ^(15)N can be between −6‰ and −7‰. These biomass values are assumed to be relatively invariant and to reflect the cellular level expressed isotope effect of nitrogen fixation. However, field and laboratory studies report wide ranges of diazotrophic biomass δ^(15)N (from −3.6‰ to +0.5‰ for Mo-based nitrogen fixation). This variation could be partly explained by the release of dissolved organic N (DON) that is isotopically distinct from biomass. The model nitrogen fixer Azotobacter vinelandii secretes siderophores, small molecules that aid in Fe uptake and can comprise >30% of fixed nitrogen. To test whether siderophores (and other released N) can decouple biomass δ^(15)N from the isotope effect of nitrogen fixation we measured the isotopic composition of biomass and released N in Fe-limited A. vinelandii cultures fixing nitrogen with Mo- and V-nitrogenases. We report that biomass δ^(15)N was elevated under Fe limitation with a maximum value of +1.2‰ for Mo-based nitrogen fixation. Regardless of the nitrogenase isozyme used, released nitrogen δ^(15)N was also 2–3‰ lower than biomass δ^(15)N. Siderophore nitrogen was found to have a slightly higher δ^(15)N than the rest of the DON pool but was still produced in large enough concentrations to account for increases in biomass δ15N. The low δ^(15)N of siderophores (relative to biomass) is consistent with what is known from compound specific isotope studies of the amino acids used in siderophore biosynthesis, and indicates that other amino-acid derived siderophores should also have a low δ^(15)N. The implications for studies of nitrogen fixation are discussed.

Phenazines liberate phosphate Bacteria secrete a wide range of small molecules with chemical reactivity that offers multiple functions in different contexts. Phenazines are commonly considered to be antibiotics, but they can also participate in environmental redox reactions, especially with iron. McRose and Newman found that phenazines, when added exogenously or made by bacteria in situ, can liberate phosphorous (P) in the form of phosphate from mineral surfaces, and that the production of these molecules is regulated by signaling pathways that respond to P limitation. Strains unable to produce these molecules grew more slowly under P limitation but could be rescued by the addition of exogenous phenazines. The authors hypothesize that reductive dissolution of iron oxides has the benefit of liberating P, and that this could be one mechanism of microbial P acquisition in some environments. Science , this issue p. 1033

The role of microbes in sustaining agricultural plant growth has great potential consequences for human prosperity. Yet we have an incomplete understanding of the basic function of rhizosphere microbial communities and how they may change under future stresses, let alone how these processes might be harnessed to sustain or improve crop yields. A reductionist approach may aid the generation and testing of hypotheses that can ultimately be translated to agricultural practices. With this in mind, we ask whether some rhizosphere microbial communities might be governed by ‘keystone metabolites’, envisioned here as microbially produced molecules that, through antibiotic and/or growth-promoting properties, may play an outsized role in shaping the development of the community spatiotemporally. To illustrate this point, we use the example of redox-active metabolites, and in particular phenazines, which are produced by many bacteria found in agricultural soils and have well-understood catalytic properties. Phenazines can act as potent antibiotics against a variety of cell types, yet they also can promote the acquisition of essential inorganic nutrients. In this essay, we suggest the ways these metabolites might affect microbial communities and ultimately agricultural productivity in two specific scenarios: firstly, in the biocontrol of beneficial and pathogenic fungi in increasingly arid crop soils and, secondly, through promotion of phosphorus bioavailability and sustainable fertilizer use. We conclude with specific proposals for future research.

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.

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 (13eMo = 13.1–14.7 ‰, 13eV = 7.5–8.8 ‰, 13eFe = 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.

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.

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 amphi-enterobactin 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.

Azotobacter vinelandii is a terrestrial diazotroph well studied for its siderophore production capacity and its role as a model nitrogen fixer. In addition to Fe, A. vinelandii siderophores are used for the acquisition of the nitrogenase co-factors Mo and V. However, regulation of siderophore production by Mo- and V-limitation has been difficult to confirm and knowledge of the full suite of siderophores synthesized by this organism has only recently become available. Using this new information, we conducted an extensive study of siderophore production in N2 -fixing A. vinelandii under a variety of trace metal conditions. Our results show that under Fe-limitation the production of all siderophores increases, while under Mo-limitation only catechol siderophore production is increased, with the strongest response seen in protochelin. We also find that the newly discovered A. vinelandii siderophore vibrioferrin is almost completely repressed under Mo- and V-limitation. An examination of the potential nitrogen 'cost' of siderophore production reveals that investments in siderophore N can represent as much as 35% of fixed N, with substantial differences between cultures using the Mo- as opposed to the less efficient V-nitrogenase.

The nitrogenase enzyme, which catalyzes the reduction of N2 gas to NH4+, occurs as three separate isoenzymes that use Mo, Fe-only, or V. The majority of global nitrogen fixation is attributed to the more efficient ‘canonical’ Mo-nitrogenase, whereas Fe-only and V-(‘alternative’) nitrogenases are often considered ‘backup’ enzymes, used when Mo is limiting. Yet, the environmental distribution and diversity of alternative nitrogenases remains largely unknown. We searched for alternative nitrogenase genes in sequenced genomes and used PacBio sequencing to explore the diversity of canonical (nifD) and alternative (anfD and vnfD) nitrogenase amplicons in two coastal environments: the Florida Everglades and Sippewissett Marsh (MA). Genome-based searches identified an additional 25 species and 10 genera not previously known to encode alternative nitrogenases. Alternative nitrogenase amplicons were found in both Sippewissett Marsh and the Florida Everglades and their activity was further confirmed using newly developed isotopic techniques. Conserved amino acid sequences corresponding to cofactor ligands were also analyzed in anfD and vnfD amplicons, offering insight into environmental variants of these motifs. This study increases the number of available anfD and vnfD sequences ~20-fold and allows for the first comparisons of environmental Mo-, Fe-only, and V-nitrogenase diversity. Our results suggest that alternative nitrogenases are maintained across a range of organisms and environments and that they can make important contributions to nitrogenase diversity and nitrogen fixation.

Cryptogamic species and their associated cyanobacteria have attracted the attention of biogeochemists 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.

Cryptophyte and chlorarachniophyte algae are transitional forms in the widespread secondary endosymbiotic acquisition of photosynthesis by engulfment of eukaryotic algae. Unlike most secondary plastid-bearing algae, miniaturized versions of the endosymbiont nuclei (nucleomorphs) persist in cryptophytes and chlorarachniophytes. To determine why, and to address other fundamental questions about eukaryote–eukaryote endosymbiosis, we sequenced the nuclear genomes of the cryptophyte Guillardia theta and the chlorarachniophyte Bigelowiella natans. Both genomes have

Summary Eukaryotic phytoplankton play key roles in atmospheric CO 2 uptake and sequestration in marine environments [1,2]. Community shifts attributed to climate change have already been reported in the Arctic ocean, where tiny, photosynthetic picoeukaryotes (≤3 μm diameter) have increased, while larger taxa have decreased [3]. Unfortunately, for vast regions of the world's oceans, little is known about distributions of different genera and levels of genetic variation between ocean basins. This lack of baseline information makes it impossible to assess the impacts of environmental change on phytoplankton diversity, and global carbon cycling. A major knowledge impediment is that these organisms are highly diverse, and most remain uncultured [2]. Metagenomics avoids the culturing step and provides insights into genes present in the environment without some of the biases associated with conventional molecular survey methods. However, connecting metagenomic sequences to the organisms containing them is challenging. For many unicellular eukaryotes the reference genomes needed to make this connection are not available. We circumvented this problem using at-sea fluorescence activated cell sorting (FACS) to separate abundant natural populations of photosynthetic eukaryotes and sequence their DNA, generating reference genome information while eliminating the need for culturing [2]. Here, we present the complete chloroplast genome from an Atlantic picoeukaryote population and discoveries it enabled on the evolution, distribution, and potential carbon sequestration role of a tiny, wild alga.

As scientists discover more about the genomes of marine microorganisms, new views of their physiology and ecosystem networks are opening up, explain Alexandra Z. Worden and Darcy McRose.