A Champion for Chemistry: Elements of Jim Morgan's Intellectual Legacy.

James (Jim) Morgan was a leading figure in the field of environmental science and engineering. He championed the application of chemistry in the study of the environment and the design and optimization of environmental treatment processes. Jim influenced the field through his classic text Aquatic Chemistry, coauthored with Werner Stumm, his role as founding editor of Environmental Science& Technology, his seminal contribution to computational methods for the calculation of chemical equilibria, and most especially, his mentorship of his extended academic family. Jim transmitted his enthusiasm for research, particularly on the chemistry of manganese and iron, so successfully to his doctoral students and postdoctoral advisees that many of them carried these topics forward in their own careers.

H-Aquil: a chemically defined cell culture medium for trace metal studies in Vibrios and other marine heterotrophic bacteria.

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.

Genetic, structural, and functional diversity of low and high-affinity siderophores in strains of nitrogen fixing Azotobacter chroococcum.

To increase iron (Fe) bioavailability in surface soils, microbes secrete siderophores, chelators with widely varying Fe affinities. Strains of the soil bacterium Azotobacter chroococcum (AC), plant-growth promoting rhizobacteria used as agricultural inoculants, require high Fe concentrations for aerobic respiration and nitrogen fixation. Recently, A. chroococcum str. NCIMB 8003 was shown to synthesize three siderophore classes: (1) vibrioferrin, a low-affinity α-hydroxy carboxylate (pFe = 18.4), (2) amphibactins, high-affinity tris-hydroxamates, and (3) crochelin A, a high-affinity siderophore with mixed Fe-chelating groups (pFe = 23.9). The relevance and specific functions of these siderophores in AC strains remain unclear. We analyzed the genome and siderophores of a second AC strain, A. chroococcum str. B3, and found that it also produces vibrioferrin and amphibactins, but not crochelin A. Genome comparisons indicate that vibrioferrin production is a vertically inherited, conserved strategy for Fe uptake in A. chroococcum and other species of Azotobacter. Amphibactin and crochelin biosynthesis reflects a more complex evolutionary history, shaped by vertical gene transfer, gene gain and loss through recombination at a genomic hotspot. We found conserved patterns of low vs. high-affinity siderophore production across strains: the low-affinity vibrioferrin was produced by mildly Fe limited cultures. As cells became more severely Fe starved, vibrioferrin production decreased in favor of high-affinity amphibactins (str. B3, NCIMB 8003) and crochelin A (str. NCIMB 8003). Our results show the evolution of low and high-affinity siderophore families and conserved patterns for their production in response to Fe bioavailability in a common soil diazotroph.

Multiple siderophores: bug or feature?

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.

Quorum sensing and iron regulate a two-for-one siderophore gene cluster in Vibrio harveyi.

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.

The purple non-sulfur bacterium Rhodopseudomonas palustris produces novel petrobactin-related siderophores under aerobic and anaerobic conditions.

Many bacteria produce siderophores to bind and take up Fe(III), an essential trace metal with extremely low solubility in oxygenated environments at circumneutral pH. The purple non-sulfur bacterium Rhodopseudomonas palustris str. CGA009 is a metabolically versatile model organism with high iron requirements that is able to grow under aerobic and anaerobic conditions. Siderophore biosynthesis has been predicted by genomic analysis, however, siderophore structures were not identified. Here, we elucidate the structure of two novel siderophores from R. palustris: rhodopetrobactin A and B. Rhodopetrobactins are structural analogues of the known siderophore petrobactin in which the Fe chelating moieties are conserved, including two 3,4-dihydroxybenzoate and a citrate substructure. In the place of two spermidine linker groups in petrobactin, rhodopetrobactins contain two 4,4'-diaminodibutylamine groups of which one or both are acetylated at the central amine. We analyse siderophore production under different growth modes and show that rhodopetrobactins are produced in response to Fe limitation under aerobic as well as under anaerobic conditions. Evaluation of the chemical characteristics of rhodopetrobactins indicates that they are well suited to support Fe acquisition under variable oxygen and light conditions.

Crochelins: Siderophores with an Unprecedented Iron-Chelating Moiety from the Nitrogen-Fixing Bacterium Azotobacter chroococcum.

Microbes use siderophores to access essential iron resources in the environment. Over 500 siderophores are known, but they utilize a small set of common moieties to bind iron. Azotobacter chroococcum expresses iron-rich nitrogenases, with which it reduces N2 . Though an important agricultural inoculant, the structures of its iron-binding molecules remain unknown. Here, the "chelome" of A. chroococcum is examined using small molecule discovery and bioinformatics. The bacterium produces vibrioferrin and amphibactins as well as a novel family of siderophores, the crochelins. Detailed characterization shows that the most abundant member, crochelin A, binds iron in a hexadentate fashion using a new iron-chelating γ-amino acid. Insights into the biosynthesis of crochelins and the mechanism by which iron may be removed upon import of the holo-siderophore are presented. This work expands the repertoire of iron-chelating moieties in microbial siderophores.

Response to Comment on "The complex effects of ocean acidification on the prominent N2-fixing cyanobacterium Trichodesmium".

Hutchins et al question the validity of our results showing that under fast growth conditions, the beneficial effect of high CO2 on Trichodesmium is overwhelmed by the deleterious effect of the concomitant decrease in ambient and cellular pH. The positive effect of acidification reported by Hutchins and co-workers is likely caused by culture conditions that support suboptimal growth rates.

Siderophore production in Azotobacter vinelandii in response to Fe-, Mo- and V-limitation.

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 complex effects of ocean acidification on the prominent N2-fixing cyanobacterium Trichodesmium.

Acidification of seawater caused by anthropogenic carbon dioxide (CO2) is anticipated to influence the growth of dinitrogen (N2)-fixing phytoplankton, which contribute a large fraction of primary production in the tropical and subtropical ocean. We found that growth and N2-fixation of the ubiquitous cyanobacterium Trichodesmium decreased under acidified conditions, notwithstanding a beneficial effect of high CO2 Acidification resulted in low cytosolic pH and reduced N2-fixation rates despite elevated nitrogenase concentrations. Low cytosolic pH required increased proton pumping across the thylakoid membrane and elevated adenosine triphosphate production. These requirements were not satisfied under field or experimental iron-limiting conditions, which greatly amplified the negative effect of acidification.

Diversity and Activity of Alternative Nitrogenases in Sequenced Genomes and Coastal Environments.

The nitrogenase enzyme, which catalyzes the reduction of N2 gas to NH4+, occurs as three separate isozyme 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.

The effects of pH and pCO2 on photosynthesis and respiration in the diatom Thalassiosira weissflogii.

The response of marine phytoplankton to the ongoing increase in atmospheric pCO2 reflects the consequences of both increased CO2 concentration and decreased pH in surface seawater. In the model diatom Thalassiosira weissflogii, we explored the effects of varying pCO2 and pH, independently and in concert, on photosynthesis and respiration by incubating samples in water enriched in H218O. In long-term experiments (~6-h) at saturating light intensity, we observed no effects of pH or pCO2 on growth rate, photosynthesis or respiration. This absence of a measurable response reflects the very small change in energy used by the carbon concentrating mechanism (CCM) compared to the energy used in carbon fixation. In short-term experiments (~3 min), we also observed no effects of pCO2 or pH, even under limiting light intensity. We surmise that in T. weissflogii, it is the photosynthetic production of NADPH and ATP, rather than the CO2-saturation of Rubisco that controls the rate of photosynthesis at low irradiance. In short-term experiments, we observed a slightly higher respiration rate at low pH at the onset of the dark period, possibly reflecting the energy used for exporting H+ and maintaining pH homeostasis. Based on what is known of the biochemistry of marine phytoplankton, our results are likely generalizable to other diatoms and a number of other eukaryotic species. The direct effects of ocean acidification on growth, photosynthesis and respiration in these organisms should be small over the range of atmospheric pCO2 predicted for the twenty-first century.

Rubisco Extraction and Purification from Diatoms.

This protocol describes a method to extract ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) from diatoms (Bacillariophyta) to determine catalytic performance. This protocol has been adapted from use in cyanobacteria and higher plants (Andrews, 1988; Whitney and Sharwood, 2007). First part (steps A1-A3) of the extraction provides a crude extract of Rubisco that is sufficient for carboxylation assays to measure the Michaelis constant for CO2 (KC) and the catalytic turnover rate ( kcat c ). However, the further purification steps outlined (steps B1-B4) are needed for measurements of Rubisco CO2/O2 Specificity (SC/O, [ Kane et al., 1994 ]).

Large variation in the Rubisco kinetics of diatoms reveals diversity among their carbon-concentrating mechanisms.

While marine phytoplankton rival plants in their contribution to global primary productivity, our understanding of their photosynthesis remains rudimentary. In particular, the kinetic diversity of the CO2-fixing enzyme, Rubisco, in phytoplankton remains unknown. Here we quantify the maximum rates of carboxylation (k cat (c)), oxygenation (k cat (o)), Michaelis constants (K m) for CO2 (K C) and O2 (K O), and specificity for CO2 over O2 (SC/O) for Form I Rubisco from 11 diatom species. Diatom Rubisco shows greater variation in K C (23-68 µM), SC/O (57-116mol mol(-1)), and K O (413-2032 µM) relative to plant and algal Rubisco. The broad range of K C values mostly exceed those of C4 plant Rubisco, suggesting that the strength of the carbon-concentrating mechanism (CCM) in diatoms is more diverse, and more effective than previously predicted. The measured k cat (c) for each diatom Rubisco showed less variation (2.1-3.7s(-1)), thus averting the canonical trade-off typically observed between K C and k cat (c) for plant Form I Rubisco. Uniquely, a negative relationship between K C and cellular Rubisco content was found, suggesting variation among diatom species in how they allocate their limited cellular resources between Rubisco synthesis and their CCM. The activation status of Rubisco in each diatom was low, indicating a requirement for Rubisco activase. This work highlights the need to better understand the correlative natural diversity between the Rubisco kinetics and CCM of diatoms and the underpinning mechanistic differences in catalytic chemistry among the Form I Rubisco superfamily.

The Siderophore Metabolome of Azotobacter vinelandii.

In this study, we performed a detailed characterization of the siderophore metabolome, or "chelome," of the agriculturally important and widely studied model organism Azotobacter vinelandii. Using a new high-resolution liquid chromatography-mass spectrometry (LC-MS) approach, we found over 35 metal-binding secondary metabolites, indicative of a vast chelome in A. vinelandii. These include vibrioferrin, a siderophore previously observed only in marine bacteria. Quantitative analyses of siderophore production during diazotrophic growth with different sources and availabilities of Fe showed that, under all tested conditions, vibrioferrin was present at the highest concentration of all siderophores and suggested new roles for vibrioferrin in the soil environment. Bioinformatic searches confirmed the capacity for vibrioferrin production in Azotobacter spp. and other bacteria spanning multiple phyla, habitats, and lifestyles. Moreover, our studies revealed a large number of previously unreported derivatives of all known A. vinelandii siderophores and rationalized their origins based on genomic analyses, with implications for siderophore diversity and evolution. Together, these insights provide clues as to why A. vinelandii harbors multiple siderophore biosynthesis gene clusters. Coupled with the growing evidence for alternative functions of siderophores, the vast chelome in A. vinelandii may be explained by multiple, disparate evolutionary pressures that act on siderophore production.

The effect of acidification on the bioavailability and electrochemical lability of zinc in seawater.

A poorly studied but potentially important consequence of the CO2-induced acidification of the surface ocean is a possible change in the bioavailability of trace metals, which play a critical role in the productivity and population dynamics of marine ecosystems. We report laboratory and field experiments designed to compare quantitatively the effects of acidification on the bioavailability of Zn, a metal essential to the growth of phytoplankton and on the extent of its complexation by model and natural ligands. We observed a good correspondence between the effects of pH on the rate of Zn uptake by a model diatom and the chemical lability of Zn measured by anodic stripping voltammetry (ASV). In model laboratory systems, the chemical lability and the bioavailability of Zn could either increase or decrease at low pH depending on the mix of complexing ligands. In a sample of coastal surface water, we observed similar increases in the ASV-labile and bioavailable Zn concentrations upon acidification, a result contrary to previous observations. These results, which can likely be generalized to other bioactive trace metals, mutatis mutandis, demonstrate the intricacy of the effects of ocean acidification on the chemistry and the ecology of surface seawater.This article is part of the themed issue 'Biological and climatic impacts of ocean trace element chemistry'.

Bioavailability and Electroreactivity of Zinc Complexed to Strong and Weak Organic Ligands.

Laboratory experiments have established the importance of complexation by organic ligands in determining the bioavailability of trace metals to marine phytoplankton, while electrochemical measurements with field samples have demonstrated that a large fraction of bioactive trace metals are complexed to strong organic ligands in seawater. Using the model organic ligands, EDTA and histidine, we show a quantitative correspondence between the bioavailability of Zn to the diatom Thalassiosira weissflogii, and its reduction at -1.2 V (vs Ag/AgCl) on a hanging mercury drop electrode. Equilibrium calculations and polarographic data indicate that Zn bound in inorganic complexes and the 1:1 Zn-histidine complex, but not in the 1:2 Zn-histidine complex or the Zn-EDTA complexes, is taken up by the organism and reduced at the electrode surface, confirming a previous report of the bioavailability of weak Zn complexes. Electrochemical measurements of Zn speciation in seawater do not generally reveal the presence of weak (and potentially bioavailable) complexes; but such measurements (particularly by Anodic Stripping Voltammetry) should nonetheless often provide good estimates of the bioavailable Zn concentrations. These results can likely be generalized to other bioactive divalent trace metals.

Effect of thiols, zinc, and redox conditions on Hg uptake in Shewanella oneidensis.

Mercury uptake in bacteria represents a key first step in the production and accumulation of methylmercury in biota. Previous experiments with mercury methylating bacteria have shown that Hg uptake is enhanced by some thiols, in particular cysteine, and that it is an energy-dependent process through heavy metal transporters [Schaefer et al. Environ. Sci. Technol. 2014, 48, 3007]. In this study, we examine Hg uptake in the nonmethylating facultative aerobe, Shewanella oneidensis, under both anaerobic and aerobic conditions. Our results demonstrate similar characteristics of the Hg uptake system to those of the Hg methylating strains: (1) uptake is enhanced in the presence of some thiols but not others; (2) uptake is energy dependent as evidenced by inhibition by a protonophore, and (3) uptake is inhibited by high Zn(II) concentrations. Initial cellular uptake rates in S. oneidensis were remarkably similar under aerobic and fumarate-reducing conditions. These data support a similar Hg(II) uptake mechanism within the proteobacteria of accidental Hg(II) transport through heavy metal transporters with similar rates of uptake but differences in the ability to take up Hg bound to different thiols.

Slow carboxylation of Rubisco constrains the rate of carbon fixation during Antarctic phytoplankton blooms.

High-latitude oceans are areas of high primary production despite temperatures that are often well below the thermal optima of enzymes, including the key Calvin Cycle enzyme, Ribulose 1,5 bisphosphate carboxylase oxygenase (Rubisco). We measured carbon fixation rates, protein content and Rubisco abundance and catalytic rates during an intense diatom bloom in the Western Antarctic Peninsula (WAP) and in laboratory cultures of a psychrophilic diatom (Fragilariopsis cylindrus). At -1°C, the Rubisco turnover rate, kcat (c) , was 0.4 C s(-1) per site and the half saturation constant for CO2 was 15 µM (vs c. 3 C s(-1) per site and 50 µM at 20°C). To achieve high carboxylation rates, psychrophilic diatoms increased Rubisco abundance to c. 8% of biomass (vs c. 0.6% at 20°C), along with their total protein content, resulting in a low carbon : nitrogen ratio of c. 5. In psychrophilic diatoms, Rubisco must be almost fully active and near CO2 saturation to achieve carbon fixation rates observed in the WAP. Correspondingly, total protein concentrations were close to the highest ever measured in phytoplankton and likely near the maximum possible. We hypothesize that this high protein concentration, like that of Rubisco, is necessitated by slow enzyme rates, and that carbon fixation rates in the WAP are near a theoretical maximum.

Iron bioavailability to phytoplankton: an empirical approach.

Phytoplankton are often limited by iron in aquatic environments. Here we examine Fe bioavailability to phytoplankton by analyzing iron uptake from various Fe substrates by several species of phytoplankton grown under conditions of Fe limitation and comparing the measured uptake rate constants (Fe uptake rate/ substrate concentration). When unchelated iron, Fe', buffered by an excess of the chelating agent EDTA is used as the Fe substrate, the uptake rate constants of all the eukaryotic phytoplankton species are tightly correlated and proportional to their respective surface areas (S.A.). The same is true when FeDFB is the substrate, but the corresponding uptake constants are one thousand times smaller than for Fe'. The uptake rate constants for the other substrates we examined fall mostly between the values for Fe' and FeDFB for the same S.A. These two model substrates thus empirically define a bioavailability envelope with Fe' at the upper and FeDFB at the lower limit of iron bioavailability. This envelope provides a convenient framework to compare the relative bioavailabilities of various Fe substrates to eukaryotic phytoplankton and the Fe uptake abilities of different phytoplankton species. Compared with eukaryotic species, cyanobacteria have similar uptake constants for Fe' but lower ones for FeDFB. The unique relationship between the uptake rate constants and the S.A. of phytoplankton species suggests that the uptake rate constant of Fe-limited phytoplankton has reached a universal upper limit and provides insight into the underlying uptake mechanism.