Helen W. Kreuzer

Mutations in Global Regulators Lead to Metabolic Selection during Adaptation to Complex Environments

Adaptation to ecologically complex environments can provide insights into the evolutionary dynamics and functional constraints encountered by organisms during natural selection. Adaptation to a new environment with abundant and varied resources can be difficult to achieve by small incremental changes if many mutations are required to achieve even modest gains in fitness. Since changing complex environments are quite common in nature, we investigated how such an epistatic bottleneck can be avoided to allow rapid adaptation. We show that adaptive mutations arise repeatedly in independently evolved populations in the context of greatly increased genetic and phenotypic diversity. We go on to show that weak selection requiring substantial metabolic reprogramming can be readily achieved by mutations in the global response regulator arcA and the stress response regulator rpoS. We identified 46 unique single-nucleotide variants of arcA and 18 mutations in rpoS, nine of which resulted in stop codons or large deletions, suggesting that subtle modulations of ArcA function and knockouts of rpoS are largely responsible for the metabolic shifts leading to adaptation. These mutations allow a higher order metabolic selection that eliminates epistatic bottlenecks, which could occur when many changes would be required. Proteomic and carbohydrate analysis of adapting E. coli populations revealed an up-regulation of enzymes associated with the TCA cycle and amino acid metabolism, and an increase in the secretion of putrescine. The overall effect of adaptation across populations is to redirect and efficiently utilize uptake and catabolism of abundant amino acids. Concomitantly, there is a pronounced spread of more ecologically limited strains that results from specialization through metabolic erosion. Remarkably, the global regulators arcA and rpoS can provide a “one-step” mechanism of adaptation to a novel environment, which highlights the importance of global resource management as a powerful strategy to adaptation.

Synchrotron based infrared imaging and spectroscopy via focal plane array on live fibroblasts in D2O enriched medium

We successfully tested the viability of using synchrotron-based full-field infrared imaging to study biochemical processes inside living cells. As a model system, we studied fibroblast cells exposed to a medium highly enriched with D2O. We could show that the experimental technique allows us to reproduce at the cellular level measurements that are normally performed on purified biological molecules. We can obtain information about lipid conformation and distribution, kinetics of hydrogen/deuterium exchange, and the formation of concentration gradients of H and O isotopes in water that are associated with cell metabolism. The implementation of the full field technique in a sequential imaging format gives a description of cellular biochemistry and biophysics that contains both spatial and temporal information.

Detection of Metabolic Fluxes of O and H Atoms into Intracellular Water in Mammalian Cells

Metabolic processes result in the release and exchange of H and O atoms from organic material as well as some inorganic salts and gases. These fluxes of H and O atoms into intracellular water result in an isotopic gradient that can be measured experimentally. Using isotope ratio mass spectroscopy, we revealed that slightly over 50% of the H and O atoms in the intracellular water of exponentially-growing cultured Rat-1 fibroblasts were isotopically distinct from growth medium water. We then employed infrared spectromicroscopy to detect in real time the flux of H atoms in these same cells. Importantly, both of these techniques indicate that the H and O fluxes are dependent on metabolic processes; cells that are in lag phase or are quiescent exhibit a much smaller flux. In addition, water extracted from the muscle tissue of rats contained a population of H and O atoms that were isotopically distinct from body water, consistent with the results obtained using the cultured Rat-1 fibroblasts. Together these data demonstrate that metabolic processes produce fluxes of H and O atoms into intracellular water, and that these fluxes can be detected and measured in both cultured mammalian cells and in mammalian tissue.

Carbon Dioxide Fixation by Metallosphaera yellowstonensis and Acidothermophilic Iron-Oxidizing Microbial Communities from Yellowstone National Park

ABSTRACT The fixation of inorganic carbon has been documented in all three domains of life and results in the biosynthesis of diverse organic compounds that support heterotrophic organisms. The primary aim of this study was to assess carbon dioxide fixation in high-temperature Fe(III)-oxide mat communities and in pure cultures of a dominant Fe(II)-oxidizing organism (Metallosphaera yellowstonensis strain MK1) originally isolated from these environments. Protein-encoding genes of the complete 3-hydroxypropionate/4-hydroxybutyrate (3-HP/4-HB) carbon dioxide fixation pathway were identified in M. yellowstonensis strain MK1. Highly similar M. yellowstonensis genes for this pathway were identified in metagenomes of replicate Fe(III)-oxide mats, as were genes for the reductive tricarboxylic acid cycle from Hydrogenobaculum spp. (Aquificales). Stable-isotope (13CO2) labeling demonstrated CO2 fixation by M. yellowstonensis strain MK1 and in ex situ assays containing live Fe(III)-oxide microbial mats. The results showed that strain MK1 fixes CO2 with a fractionation factor of ∼2.5‰. Analysis of the 13C composition of dissolved inorganic C (DIC), dissolved organic C (DOC), landscape C, and microbial mat C showed that mat C is from both DIC and non-DIC sources. An isotopic mixing model showed that biomass C contains a minimum of 42% C of DIC origin, depending on the fraction of landscape C that is present. The significance of DIC as a major carbon source for Fe(III)-oxide mat communities provides a foundation for examining microbial interactions that are dependent on the activity of autotrophic organisms (i.e., Hydrogenobaculum and Metallosphaera spp.) in simplified natural communities.

Approaches to Plant Hydrogen and Oxygen Isoscapes Generation

Our understanding of the δ2H and δ18O of plant water and tissues ranges from model descriptions of multiple fractionating processes and mechanistic drivers, to primarily observational relationships often described by simple regression relationships. Plant hydrogen and oxygen isoscapes are therefore produced with a range of model sophistications and demonstrate some of the diversity of approaches to producing and utilizing isoscapes in general. Leaf water is central to much of plant H & O isoscape modeling. It affects the atmosphere at large scales by influencing atmospheric CO2 and O2 δ18O, and the biosphere at a range of scales through its influence on the isotopic composition of plant organic compounds. Leaf water isoscapes have therefore been produced as part of efforts to understand atmospheric gas isotopic composition and also have the potential to contribute to other areas where spatial variation in plant-derived organic compound isotope ratios is of interest. For example, improving our understanding of the isotope ratios of cellulose or n-alkanes improves their utility for paleoreconstructions. Spatially-explicit models of food isotope ratios may improve the use of animal isotope ratios to infer migration patterns. Finally, plant hydrogen and oxygen isoscapes can yield forensic information for a variety of products, including food, drugs, or other plant-derived materials. Given this wide range of applications, we discuss approaches to producing plant H & O isoscapes that vary in their complexity depending on the level of mechanistic understanding, data availability, and the question being pursued.

Laser ablation isotope ratio mass spectrometry for enhanced sensitivity and spatial resolution in stable isotope analysis

Stable isotope analysis permits the tracking of physical, chemical, and biological reactions and source materials at a wide variety of spatial scales. We present a laser ablation isotope ratio mass spectrometry (LA-IRMS) method that enables δ(13)C measurement of solid samples at 50 µm spatial resolution. The method does not require sample pre-treatment to physically separate spatial zones. We use laser ablation of solid samples followed by quantitative combustion of the ablated particulates to convert sample carbon into CO(2). Cryofocusing of the resulting CO(2) coupled with modulation in the carrier flow rate permits coherent peak introduction into an isotope ratio mass spectrometer, with only 65 ng carbon required per measurement. We conclusively demonstrate that the measured CO(2) is produced by combustion of laser-ablated aerosols from the sample surface. We measured δ(13)C for a series of solid compounds using laser ablation and traditional solid sample analysis techniques. Both techniques produced consistent isotopic results but the laser ablation method required over two orders of magnitude less sample. We demonstrated that LA-IRMS sensitivity coupled with its 50 µm spatial resolution could be used to measure δ(13) C values along a length of hair, making multiple sample measurements over distances corresponding to a single days growth. This method will be highly valuable in cases where the δ(13)C analysis of small samples over prescribed spatial distances is required. Suitable applications include forensic analysis of hair samples, investigations of tightly woven microbial systems, and cases of surface analysis where there is a sharp delineation between different components of a sample.

Stable Carbon and Nitrogen Isotope Ratios of Sodium and Potassium Cyanide as a Forensic Signature

Abstract:  Sodium and potassium cyanide are highly toxic, produced in large amounts by the chemical industry, and linked to numerous high‐profile crimes. The U.S. Centers for Disease Control and Prevention has identified cyanide as one of the most probable agents to be used in a chemical terrorism event. We investigated whether stable C and N isotopic content of sodium and potassium cyanide could serve as a forensic signature for sample matching, using a collection of 65 cyanide samples. Upon analysis, a few of the cyanide samples displayed nonhomogeneous isotopic content associated with degradation to a carbonate salt and loss of hydrogen cyanide. Most samples had highly reproducible isotope content. Of the 65 cyanide samples, >95% could be properly matched based on C and N isotope ratios, with a false match rate <3%. These results suggest that stable C and N isotope ratios are a useful forensic signature for matching cyanide samples.

Analysis of carbohydrate and fatty acid marker abundance in ricin toxin preparations for forensic information.

One challenge in the forensic analysis of ricin samples is determining the method and extent of sample preparation. Ricin purification from the source castor seeds is essentially a protein purification through removal of the nonprotein fractions of the seed. Two major, nonprotein constituents in the seed are the castor oil and carbohydrates. We used derivatization of carbohydrate and fatty acid markers followed by identification and quantification using gas chromatography/mass spectrometry (GC/MS) to assess compositional changes in ricin samples purified by different methods. The loss of ricinoleic acid indicated steps for oil removal had occurred, and a large decrease of ricinoleic acid was observed between unextracted mash and solvent extracted and protein precipitate preparations. Changes to the carbohydrate content of the sample were also observed following protein precipitation. The differential loss of arabinose relative to mannose was observed indicating the removal of the major carbohydrate fraction of the seed and enrichment of the protein content. When the data is combined and multivariate principle component analysis is applied, these changes in fatty acid and carbohydrate abundance are discriminating enough to be indicative of the preparation method used for each sample.

Using gas chromatography/isotope ratio mass spectrometry to determine the fractionation factor for H2 production by hydrogenases

Hydrogenases catalyze the reversible formation of H(2), and they are key enzymes in the biological cycling of H(2). H isotopes have the potential to be a very useful tool in quantifying hydrogen ion trafficking in biological H(2) production processes, but there are several obstacles that have thus far limited the application of this tool. Here, we describe a new method that overcomes some of these barriers and is specifically designed to measure isotopic fractionation during enzyme-catalyzed H(2) evolution. A key feature of this technique is that purified hydrogenases are employed, allowing precise control over the reaction conditions and therefore a high level of precision. In addition, a custom-designed high-throughput gas chromatograph/isotope ratio mass spectrometer is employed to measure the isotope ratio of the H(2). Using our new approach, we determined that the fractionation factor for H(2) production by the [NiFe]-hydrogenase from Desulfovibrio fructosovorans is 0.273 ± 0.006. This result indicates that, as expected, protons are highly favored over deuterium ions during H(2) evolution. Potential applications of this newly developed method are discussed.

Detection of Acetone Processing of Castor Bean Mash for Forensic Investigation of Ricin Preparation Methods

Abstract:  Samples containing the toxic castor bean protein ricin have been recently seized in connection with biocriminal activity. Analytical methods that enable investigators to determine how the samples were prepared and to match seized samples to potential source materials are needed. One commonly described crude ricin preparation method is acetone extraction of crushed castor beans. Here, we describe the use of solid‐phase microextraction and headspace analysis to determine whether castor beans were processed by acetone extraction. We prepared acetone‐extracted castor bean mash, along with controls of unextracted mash and mash extracted with nonacetone organic solvents. Samples of acetone‐extracted mash and unextracted mash were stored in closed containers for up to 109 days at both room temperature and −20°C, and in open containers at room temperature for up to 94 days. Acetone‐extracted bean mash could consistently be statistically distinguished from controls, even after storage in open containers for 94 days.