Jessica R. Gooding

Riboneogenesis in yeast.

Glucose is catabolized in yeast via two fundamental routes, glycolysis and the oxidative pentose phosphate pathway, which produces NADPH and the essential nucleotide component ribose-5-phosphate. Here, we describe riboneogenesis, a thermodynamically driven pathway that converts glycolytic intermediates into ribose-5-phosphate without production of NADPH. Riboneogenesis begins with synthesis, by the combined action of transketolase and aldolase, of the seven-carbon bisphosphorylated sugar sedoheptulose-1,7-bisphosphate. In the pathways committed step, sedoheptulose bisphosphate is hydrolyzed to sedoheptulose-7-phosphate by the enzyme sedoheptulose-1,7-bisphosphatase (SHB17), whose activity we identified based on metabolomic analysis of the corresponding knockout strain. The crystal structure of Shb17 in complex with sedoheptulose-1,7-bisphosphate reveals that the substrate binds in the closed furan form in the active site. Sedoheptulose-7-phosphate is ultimately converted by known enzymes of the nonoxidative pentose phosphate pathway to ribose-5-phosphate. Flux through SHB17 increases when ribose demand is high relative to demand for NADPH, including during ribosome biogenesis in metabolically synchronized yeast cells.

Transcriptomic and metabolomic profiling of chicken adipose tissue in response to insulin neutralization and fasting

BackgroundDomestic broiler chickens rapidly accumulate adipose tissue due to intensive genetic selection for rapid growth and are naturally hyperglycemic and insulin resistant, making them an attractive addition to the suite of rodent models used for studies of obesity and type 2 diabetes in humans. Furthermore, chicken adipose tissue is considered as poorly sensitive to insulin and lipolysis is under glucagon control. Excessive fat accumulation is also an economic and environmental concern for the broiler industry due to the loss of feed efficiency and excessive nitrogen wasting, as well as a negative trait for consumers who are increasingly conscious of dietary fat intake. Understanding the control of avian adipose tissue metabolism would both enhance the utility of chicken as a model organism for human obesity and insulin resistance and highlight new approaches to reduce fat deposition in commercial chickens.ResultsWe combined transcriptomics and metabolomics to characterize the response of chicken adipose tissue to two energy manipulations, fasting and insulin deprivation in the fed state. Sixteen to 17 day-old commercial broiler chickens (ISA915) were fed ad libitum, fasted for five hours, or fed but deprived of insulin by injections of anti-insulin serum. Pair-wise contrasts of expression data identified a total of 2016 genes that were differentially expressed after correction for multiple testing, with the vast majority of differences due to fasting (1780 genes). Gene Ontology and KEGG pathway analyses indicated that a short term fast impacted expression of genes in a broad selection of pathways related to metabolism, signaling and adipogenesis. The effects of insulin neutralization largely overlapped with the response to fasting, but with more modest effects on adipose tissue metabolism. Tissue metabolomics indicated unique effects of insulin on amino acid metabolism.ConclusionsCollectively, these data provide a foundation for further study into the molecular basis for adipose expansion in commercial poultry and identify potential pathways through which fat accretion may be attenuated in the future through genetic selection or management practices. They also highlight chicken as a useful model organism in which to study the dynamic relationship between food intake, metabolism, and adipose tissue biology.

Phage infection of an environmentally relevant marine bacterium alters host metabolism and lysate composition

Viruses contribute to the mortality of marine microbes, consequentially altering biological species composition and system biogeochemistry. Although it is well established that host cells provide metabolic resources for virus replication, the extent to which infection reshapes host metabolism at a global level and the effect of this alteration on the cellular material released following viral lysis is less understood. To address this knowledge gap, the growth dynamics, metabolism and extracellular lysate of roseophage-infected Sulfitobacter sp. 2047 was studied using a variety of techniques, including liquid chromatography–tandem mass spectrometry (LC-MS/MS)-based metabolomics. Quantitative estimates of the total amount of carbon and nitrogen sequestered into particulate biomass indicate that phage infection redirects ∼75% of nutrients into virions. Intracellular concentrations for 82 metabolites were measured at seven time points over the infection cycle. By the end of this period, 71% of the detected metabolites were significantly elevated in infected populations, and stable isotope-based flux measurements showed that these cells had elevated metabolic activity. In contrast to simple hypothetical models that assume that extracellular compounds increase because of lysis, a profile of metabolites from infected cultures showed that >70% of the 56 quantified compounds had decreased concentrations in the lysate relative to uninfected controls, suggesting that these small, labile nutrients were being utilized by surviving cells. These results indicate that virus-infected cells are physiologically distinct from their uninfected counterparts, which has implications for microbial community ecology and biogeochemistry.

Two-dimensional liquid chromatography/mass spectrometry/mass spectrometry separation of water soluble metabolites:

Off-line two-dimensional liquid chromatography with tandem mass spectrometry detection (2D-LC/MS-MS) was used to separate a set of metabolomic species. Water-soluble metabolites were extracted from Escherichia coli and Saccharomyces cerevisae cultures and were immediately analyzed using strong cation exchange (SCX)-hydrophilic interaction chromatography (HILIC). Metabolite mixtures are well-suited for multidimensional chromatography as the range of components varies widely with respect to polarity and chemical makeup. Some currently used methods employ two different separations for the detection of positively and negatively ionized metabolites by mass spectrometry. Here we developed a single set of chromatographic conditions for both ionization modes and were able to detect a total of 141 extracted metabolite species, with an overall peak capacity of ca. 2500. We show that a single two-dimensional separation method is sufficient and practical when a pair or more of unidimensional separations are used in metabolomics.

Integration of the Second Messenger c-di-GMP into the Chemotactic Signaling Pathway

ABSTRACT Elevated intracellular levels of the bacterial second messenger c-di-GMP are known to suppress motility and promote sessility. Bacterial chemotaxis guides motile cells in gradients of attractants and repellents over broad concentration ranges, thus allowing bacteria to quickly adapt to changes in their surroundings. Here, we describe a chemotaxis receptor that enhances, as opposed to suppresses, motility in response to temporary increases in intracellular c-di-GMP. Azospirillum brasilense’s preferred metabolism is adapted to microaerophily, and these motile cells quickly navigate to zones of low oxygen concentration by aerotaxis. We observed that changes in oxygen concentration result in rapid changes in intracellular c-di-GMP levels. The aerotaxis and chemotaxis receptor, Tlp1, binds c-di-GMP via its C-terminal PilZ domain and promotes persistent motility by increasing swimming velocity and decreasing swimming reversal frequency, which helps A. brasilense reach low-oxygen zones. If c-di-GMP levels remain high for extended periods, A. brasilense forms nonmotile clumps or biofilms on abiotic surfaces. These results suggest that association of increased c-di-GMP levels with sessility is correct on a long-term scale, while in the short-term c-di-GMP may actually promote, as opposed to suppress, motility. Our data suggest that sensing c-di-GMP by Tlp1 functions similar to methylation-based adaptation. Numerous chemotaxis receptors contain C-terminal PilZ domains or other sensory domains, suggesting that intracellular c-di-GMP as well as additional stimuli can be used to modulate adaptation of bacterial chemotaxis receptors. IMPORTANCE To adapt and compete under changing conditions, bacteria must not only detect and respond to various environmental cues but also be able to remain sensitive to further changes in the environmental conditions. In bacterial chemotaxis, chemosensory sensitivity is typically brought about by changes in the methylation status of chemotaxis receptors capable of modulating the ability of motile cells to navigate in gradients of various physicochemical cues. Here, we show that the ubiquitous second messenger c-di-GMP functions to modulate chemosensory sensitivity of a bacterial chemotaxis receptor in the alphaproteobacterium Azospirillum brasilense. Binding of c-di-GMP to the chemotaxis receptor promotes motility under conditions of elevated intracellular c-di-GMP levels. Our results revealed that the role of c-di-GMP as a sessile signal is overly simplistic. We also show that adaptation by sensing an intracellular metabolic cue, via PilZ or other domains, is likely widespread among bacterial chemotaxis receptors. To adapt and compete under changing conditions, bacteria must not only detect and respond to various environmental cues but also be able to remain sensitive to further changes in the environmental conditions. In bacterial chemotaxis, chemosensory sensitivity is typically brought about by changes in the methylation status of chemotaxis receptors capable of modulating the ability of motile cells to navigate in gradients of various physicochemical cues. Here, we show that the ubiquitous second messenger c-di-GMP functions to modulate chemosensory sensitivity of a bacterial chemotaxis receptor in the alphaproteobacterium Azospirillum brasilense. Binding of c-di-GMP to the chemotaxis receptor promotes motility under conditions of elevated intracellular c-di-GMP levels. Our results revealed that the role of c-di-GMP as a sessile signal is overly simplistic. We also show that adaptation by sensing an intracellular metabolic cue, via PilZ or other domains, is likely widespread among bacterial chemotaxis receptors.

Direct quantitation of the quorum sensing signal, autoinducer-2, in clinically relevant samples by liquid chromatography-tandem mass spectrometry.

Quorum Sensing is a type of bacterial cell-to-cell signaling that allows for cell density dependent regulation of gene expression. Many of the behaviors mediated by quorum sensing are critical for bacterial colonization or infection, and autoinducer-2 has been proposed as a universal interspecies signaling molecule that allows multispecies colonies of bacteria, e.g., biofilms or dental plaque, to behave as pseudomulticellular organisms. However, the direct detection of autoinducer-2 has been difficult, leaving the in vivo relevance of this signal in question. Herein we report a liquid chromatography-tandem mass spectrometric technique that enables reproducible, quantitative, and sensitive measurement of the concentration of autoinducer-2 from a variety of sources. This technique was applied to the detection of autoinducer-2 from Escherichia coli and Vibrio harveyi in proof-of-concept studies and was then used to directly measure the concentration of the signal produced by oral bacteria in human saliva.

Establishing a quantitative definition of quorum sensing provides insight into the information content of the autoinducer signals in Vibrio harveyi and Escherichia coli.

Extracellular autoinducer concentrations in cultures of Vibrio harveyi and Escherichia coli were monitored by liquid chromatography-tandem mass spectrometry to test whether a quantitative definition of quorum sensing could help decipher the information content of these signals. Although V. harveyi was able to keep the autoinducer-2 to cell number ratio constant, the ratio of signal to cell number for V. harveyi autoinducer-1 and E. coli autoinducer-2 varied as the cultures grew. These data indicate that V. harveyi uses autoinducer-2 for quorum sensing, while the other molecules may be used to transmit different information or are influenced by metabolic noise.

MetabR: an R script for linear model analysis of quantitative metabolomic data

BackgroundMetabolomics is an emerging high-throughput approach to systems biology, but data analysis tools are lacking compared to other systems level disciplines such as transcriptomics and proteomics. Metabolomic data analysis requires a normalization step to remove systematic effects of confounding variables on metabolite measurements. Current tools may not correctly normalize every metabolite when the relationships between each metabolite quantity and fixed-effect confounding variables are different, or for the effects of random-effect confounding variables. Linear mixed models, an established methodology in the microarray literature, offer a standardized and flexible approach for removing the effects of fixed- and random-effect confounding variables from metabolomic data.FindingsHere we present a simple menu-driven program, “MetabR”, designed to aid researchers with no programming background in statistical analysis of metabolomic data. Written in the open-source statistical programming language R, MetabR implements linear mixed models to normalize metabolomic data and analysis of variance (ANOVA) to test treatment differences. MetabR exports normalized data, checks statistical model assumptions, identifies differentially abundant metabolites, and produces output files to help with data interpretation. Example data are provided to illustrate normalization for common confounding variables and to demonstrate the utility of the MetabR program.ConclusionsWe developed MetabR as a simple and user-friendly tool for implementing linear mixed model-based normalization and statistical analysis of targeted metabolomic data, which helps to fill a lack of available data analysis tools in this field. The program, user guide, example data, and any future news or updates related to the program may be found athttp://metabr.r-forge.r-project.org/.

Sex- and Strain-dependent Effects of Bisphenol: A Consumption inJuvenile Mice

Children are directly exposed to bisphenol A (BPA) and other putative environmental obesogens through both diet and household products. The purpose of this study was to investigate sex- and genetic-dependent effects of BPA consumption on traits relevant to obesity and Type 2 diabetes in juveniles, using mice as models. Two strains of mice (C57BL/6J and DBA/2J) were chosen to represent distinct genetic backgrounds that differ in susceptibility to obesity and insulin resistance. Male and female mice of each strain consumed BPA at three doses in drinking water from four to eleven weeks of age. Male but not female mice of each strain consuming BPA were significantly fatter than controls. BPA induced corresponding changes in adipose tissue gene expression and metabolite abundance that indicate effects on adipogenesis and energy utilization. BPA consumption also altered the synthesis of adipokines, circulating factors that modulate insulin sensitivity and atherogenesis, in C57BL/6J but not DBA/2J males. Conversely, effects of BPA on plasma insulin were only observed in female mice, and with opposite effects in the two strains. These results suggest that dietary BPA may exacerbate childhood obesity and its consequences, and that sex and genetic background are important determinants of the physiological impact of BPA.