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.

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.

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.

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.

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.

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.

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 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.

Nitrogen isotope fractionation by alternative nitrogenases and past ocean anoxia.

Biological nitrogen fixation constitutes the main input of fixed nitrogen to Earth's ecosystems, and its isotope effect is a key parameter in isotope-based interpretations of the N cycle. The nitrogen isotopic composition (δ(15)N) of newly fixed N is currently believed to be ∼-1‰, based on measurements of organic matter from diazotrophs using molybdenum (Mo)-nitrogenases. We show that the vanadium (V)- and iron (Fe)-only "alternative" nitrogenases produce fixed N with significantly lower δ(15)N (-6 to -7‰). An important contribution of alternative nitrogenases to N2 fixation provides a simple explanation for the anomalously low δ(15)N (<-2‰) in sediments from the Cretaceous Oceanic Anoxic Events and the Archean Eon. A significant role for the alternative nitrogenases over Mo-nitrogenase is also consistent with evidence of Mo scarcity during these geologic periods, suggesting an additional dimension to the coupling between the global cycles of trace elements and nitrogen.

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.

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 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'.

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.

Gross and net production during the spring bloom along the Western Antarctic Peninsula.

This study explores some of the physiological mechanisms responsible for high productivity near the shelf in the Western Antarctic Peninsula despite a short growing season and cold temperature. We measured gross and net primary production at Palmer Station during the summer of 2012/2013 via three different techniques: incubation with H2 (18) O; incubation with (14) CO2 ; and in situ measurements of O2 /Ar and triple oxygen isotope. Additional laboratory experiments were performed with the psychrophilic diatom Fragilariopsis cylindrus. During the spring bloom, which accounted for more than half of the seasonal gross production at Palmer Station, the ratio of net-to-gross production reached a maximum greater than c. 60%, among the highest ever reported. The use of multiple techniques showed that these high ratios resulted from low heterotrophic respiration and very low daylight autotrophic respiration. Laboratory experiments revealed a similar ratio of net-to-gross O2 production in F. cylindrus and provided the first experimental evidence for an important level of cyclic electron flow (CEF) in this organism. The low ratio of community respiration to gross primary production observed during the bloom at Palmer Station may be characteristic of high latitude coastal ecosystems and partially supported by a very active CEF in psychrophilic phytoplankton.

Low temperature reduces the energetic requirement for the CO2 concentrating mechanism in diatoms.

The goal of this study is to investigate the CO2 concentrating mechanism (CCM) of the dominant phytoplankton species during the growing season at Palmer station in the Western Antarctic Peninsula. Key CCM parameters (cellular half-saturation constants for CO2 fixation, carbonic anhydrase activity, CO2 /HCO3 (-) uptake, δ(13) Corg ) in natural phytoplankton assemblages were determined. Those results, together with additional measurements on CO2 membrane permeability from Fragilariopsis cylindrus laboratory cultures, were used to develop a numerical model of the CCM of cold water diatoms. The field data demonstrate that the dominant species throughout the season possess an effective CCM, which achieves near saturation of CO2 for fixation. The model provides a means to examine the role of eCA activity and HCO3 (-) /CO2 uptake in the functioning of the CCM. According to the model, the increase in δ(13) Corg during the bloom results chiefly from decreasing ambient CO2 concentration (which reduces the gross diffusive flux across the membrane) rather than a shift in inorganic carbon uptake from CO2 to HCO3 (-) . The CCM of diatoms in the Western Antarctic Peninsula functions with a relatively small expenditure of energy, resulting chiefly from the low half-saturation constant for Rubisco at cold temperatures.

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.

Ocean acidification slows nitrogen fixation and growth in the dominant diazotroph Trichodesmium under low-iron conditions.

Dissolution of anthropogenic CO(2) increases the partial pressure of CO(2) (pCO(2)) and decreases the pH of seawater. The rate of Fe uptake by the dominant N(2)-fixing cyanobacterium Trichodesmium declines as pH decreases in metal-buffered medium. The slower Fe-uptake rate at low pH results from changes in Fe chemistry and not from a physiological response of the organism. Contrary to previous observations in nutrient-replete media, increasing pCO(2)/decreasing pH causes a decrease in the rates of N(2) fixation and growth in Trichodesmium under low-Fe conditions. This result was obtained even though the bioavailability of Fe was maintained at a constant level by increasing the total Fe concentration at low pH. Short-term experiments in which pCO(2) and pH were varied independently showed that the decrease in N(2) fixation is caused by decreasing pH rather than by increasing pCO(2) and corresponds to a lower efficiency of the nitrogenase enzyme. To compensate partially for the loss of N(2) fixation efficiency at low pH, Trichodesmium synthesizes additional nitrogenase. This increase comes partly at the cost of down-regulation of Fe-containing photosynthetic proteins. Our results show that although increasing pCO(2) often is beneficial to photosynthetic marine organisms, the concurrent decreasing pH can affect primary producers negatively. Such negative effects can occur both through chemical mechanisms, such as the bioavailability of key nutrients like Fe, and through biological mechanisms, as shown by the decrease in N(2) fixation in Fe-limited Trichodesmium.

ChelomEx: Isotope-assisted discovery of metal chelates in complex media using high-resolution LC-MS.

Chelating agents can control the speciation and reactivity of trace metals in biological, environmental, and laboratory-derived media. A large number of trace metals (including Fe, Cu, Zn, Hg, and others) show characteristic isotopic fingerprints that can be exploited for the discovery of known and unknown organic metal complexes and related chelating ligands in very complex sample matrices using high-resolution liquid chromatography mass spectrometry (LC-MS). However, there is currently no free open-source software available for this purpose. We present a novel software tool, ChelomEx, which identifies isotope pattern-matched chromatographic features associated with metal complexes along with free ligands and other related adducts in high-resolution LC-MS data. High sensitivity and exclusion of false positives are achieved by evaluation of the chromatographic coherence of the isotope pattern within chromatographic features, which we demonstrate through the analysis of bacterial culture media. A built-in graphical user interface and compound library aid in identification and efficient evaluation of results. ChelomEx is implemented in MatLab. The source code, binaries for MS Windows and MAC OS X as well as test LC-MS data are available for download at SourceForge ( http://sourceforge.net/projects/chelomex ).

Effect of divalent metals on Hg(II) uptake and methylation by bacteria.

The production of methylmercury by some bacteria is a key first step in the accumulation and biomagnification of this toxic substance in aquatic food webs, a major human health concern. By direct measurement of cellular Hg(II) uptake in model iron and sulfate reducing bacteria, we have observed that specific trace metals, such as Zn(II) and Cd(II), inhibit uptake and methylation in these organisms, whereas other metals, such as Ni(II), Co(II), or Fe(II), do not. The inhibition of Hg(II) methylation by Zn(II) was competitive in nature and related to the concentration of inorganically complexed Zn(II) (Zn'). The inhibition of Hg(II) methylation was alleviated by decreasing the free Zn' concentration through complexation with nitrilotriacetic acid without altering the speciation of Hg(II). The inhibitory effect by Zn(II) was observed when either Hg-cysteine complexes or neutral HgCl2 dominated the speciation of Hg(II), demonstrating that both charged and neutral species are transported into the cytosol by an active rather than passive process. We propose that Hg(II) uptake is the result of its accidental uptake by metal transporter(s), possibly one effecting the transport of Zn(II).