Oliver Baars

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 ).

The Siderophore Metabolome of Azotobacter vinelandii

ABSTRACT 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.

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.

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’.

Meteorology and oceanography of the Atlantic sector of the Southern Ocean—a review of German achievements from the last decade

In the early 1980s, Germany started a new era of modern Antarctic research. The Alfred Wegener Institute Helmholtz Centre for Polar and Marine Research (AWI) was founded and important research platforms such as the German permanent station in Antarctica, today called Neumayer III, and the research icebreaker Polarstern were installed. The research primarily focused on the Atlantic sector of the Southern Ocean. In parallel, the German Research Foundation (Deutsche Forschungsgemeinschaft, DFG) started a priority program ‘Antarctic Research’ (since 2003 called SPP-1158) to foster and intensify the cooperation between scientists from different German universities and the AWI as well as other institutes involved in polar research. Here, we review the main findings in meteorology and oceanography of the last decade, funded by the priority program. The paper presents field observations and modelling efforts, extending from the stratosphere to the deep ocean. The research spans a large range of temporal and spatial scales, including the interaction of both climate components. In particular, radiative processes, the interaction of the changing ozone layer with large-scale atmospheric circulations, and changes in the sea ice cover are discussed. Climate and weather forecast models provide an insight into the water cycle and the climate change signals associated with synoptic cyclones. Investigations of the atmospheric boundary layer focus on the interaction between atmosphere, sea ice and ocean in the vicinity of polynyas and leads. The chapters dedicated to polar oceanography review the interaction between the ocean and ice shelves with regard to the freshwater input and discuss the changes in water mass characteristics, ventilation and formation rates, crucial for the deepest limb of the global, climate-relevant meridional overturning circulation. They also highlight the associated storage of anthropogenic carbon as well as the cycling of carbon, nutrients and trace metals in the ocean with special emphasis on the Weddell Sea.

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

Significance Siderophores are small molecules used by microbes to acquire the essential metal Fe in environments ranging from infections to the open ocean. Although many bacteria use quorum sensing (QS) to regulate siderophore production, the resulting siderophore concentrations and their utility for Fe uptake have been underexplored. We report that Vibrio harveyi uses a “two-for-one,” QS- and Fe-repressed gene cluster to produce cell-associated siderophores at low cell density and accumulate soluble siderophores at high cell density. Fe-uptake experiments show that these soluble siderophores are used in Fe acquisition. We also find that QS repression helps prevent the accumulation of siderophore concentrations in excess of cellular uptake capacities, an explanation that may be generalizable to other bacteria. 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.

Small Molecule LC-MS/MS Fragmentation Data Analysis and Application to Siderophore Identification

Rapid developments in tandem liquid chromatography-mass spectrometry (LCMS/MS) have created wide interest in applications for the analysis of small molecule mixtures. MS/MS spectra can contain rich structural information, but because of the structural diversity of small molecules and different data acquisition methods, analysis algorithms and workflows frequently need to be tailored to individual research questions. This chapter shows how MATLAB can be used for LC-MS/MS-based structural characterization of small molecules. Starting with the import of raw data, ways for visualization and the creation of graphical user interfaces (GUIs) for individ‐ ual applications are demonstrated. A selection of frequently used algorithms for preprocessing and data analysis is reviewed in context of their MATLAB implementation. The approaches are then tailored and applied to the analysis of iron-binding peptides (peptidic siderophores) by high-resolution LC-MS/MS. The method uses a database with siderophore structures to exploit prior knowledge about siderophore structural diversity for the interpretation of MS/MS spectra from known and new siderophores.