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The ability to monitor isotopic fractionation in terrestrial ecosystems is a challenge due to the presence of interacting variables. A laboratory-scale apparatus for controlled experiments could serve as a useful platform to deconvolute the variables that affect isotopic fractionation. Such a device could offer a powerful means to understand fractionation of carbon stocks in terrestrial ecosystems and to probe the effects of photosynthesis or interactions between the soil and plants on carbon fractionation. To this end, an enclosed and artificially-lit benchtop soil and plant chamber was constructed and equipped to monitor atmospheric isotopic composition. Fourier-transform infrared (FTIR) spectroscopy was employed for isotopic sensing since it enables in-situ measurements. The validity of FTIR for isotopic ratio determination was confirmed by comparing FTIR and isotope ratio mass spectrometry data for a series of CO2 gas samples with known quantities of 13C and 12C. The greenhouse chamber was also equipped with an optically-based trace gas analyzer capable of continuously tracking CO2, CH4, and H2O concentrations and a residual gas analyzer mass spectrometer. Reflectance spectroscopy was also incorporated by way of sealed fiber optic feed-throughs coupled to a spectro-radiometer, for quantifying changes in leaf spectra induced by various environmental stressors. The resulting greenhouse chamber can be a useful tool for determining the effects of atmospheric trace gases on plant morphology and physiology as a function of concentration and isotopic composition. Microecosystems can be examined under controlled laboratory conditions and a wide variety of plant species can be accommodated. The bench-scale greenhouse should prove useful in assessing the impact of environmental variables and in guiding the design of field experiments.

The speciation of NO2, NO, and N2O gases formed from the dissolution of copper in nitric acid is measured and characterized using Fourier-transform infrared (FTIR) spectroscopy. This study describes analysis of the gas phase species formed and ingrowths of their concentrations as a function of time. Acid concentrations range from 14 to 4 M and the NO2, NO, and N2O are analyzed using FTIR between 1585 and 2209 wavenumbers. The concentrations of the gases range from 0 to 230 ppm and change over time and as a function of acid concentration.

The speciation of gases formed in the vapor phase above HNO3-H2O mixtures at room temperature is measured and characterized using Fourier-transform infrared (FTIR) spectroscopy. Although studies of speciation of compounds have been performed in the solution phase, this study focuses solely on the gas phase species formed over HNO3-H2O mixtures and their concentrations as a function of time. The nitric acid concentration in the experiments range from 14 to 4 M. The NO2, NO, and N2O (collectively known at NOx) gases are measured by FTIR at 1585, 1850, and 2208 wavenumbers, respectively. The concentrations of these NOx species ranges from 0 to 3 ppm and change over time as a function of acid concentration. Two purities of commercially available HNO3 are used to prepare the HNO3-H2O mixtures. An analysis of thermodynamic data shows that the formation of NOx gases without the presence of a catalyst are thermodynamically favorable.

An integrated Raman spectroscopy and laser-induced breakdown spectroscopy (LIBS) instrument is a valuable geoanalytical tool for future planetary missions to Mars, Venus, and elsewhere. The ChemCam instrument operating on the Mars Curiosity rover includes a remote LIBS instrument. An integrated Raman-LIBS spectrometer (RLS) based on the ChemCam architecture could be used as a reconnaissance tool for other contact instruments as well as a primary science instrument capable of quantitative mineralogical and geochemical analyses. Replacing one of the ChemCam spectrometers with a miniature transmission spectrometer enables a Raman spectroscopy mineralogical analysis to be performed, complementing the LIBS chemical analysis while retaining an overall architecture resembling ChemCam. A prototype transmission spectrometer was used to record Raman spectra under both Martian and Venus conditions. Two different high-pressure and high-temperature cells were used to collect the Raman and LIBS spectra to simulate surface conditions on Venus. The resulting LIBS spectra were used to generate a limited partial least squares Venus calibration model for the major elements. These experiments demonstrate the utility and feasibility of a combined RLS instrument.

Bacterial response regulators (RR) that function as transcription factors in two component signaling pathways are crucial for ensuring tight regulation and coordinated expression of the genome. Currently, consensus DNA binding sites in the promoter for very few bacterial RRs have been identified. A systematic method to characterize these DNA binding sites for RRs would enable prediction of specific gene expression patterns in response to extracellular stimuli. To identify RR DNA binding sites, we functionally activated RRs using beryllofluoride and applied them to a protein-binding microarray (PBM) to discover DNA binding motifs for RRs expressed in Burkholderia, a Gram-negative bacterial genus. We identified DNA binding motifs for conserved RRs in Burkholderia thailandensis, including KdpE, RisA, and NarL, as well as for a previously uncharacterized RR at locus BTH_II2335 and its ortholog in the human pathogen Burkholderia pseudomallei at locus BPSS2315. We further demonstrate RR binding of predicted genomic targets for the two orthologs using gel shift assays and reveal a pattern of RR regulation of expression of self and other two component systems. Our studies illustrate the use of PBMs to identify DNA binding specificities for bacterial RRs and enable prediction of gene regulatory networks in response to two component signaling.

Determining transcription factor (TF) recognition motifs or operator sites is central to understanding gene regulation, yet few operators have been characterized. In this study, we used a protein-binding microarray (PBM) to discover the DNA recognition sites and putative regulons for three TetR and one MarR family TFs derived from Burkholderia xenovorans, which are common to the genus Burkholderia. We also describe the development and application of a more streamlined version of the PBM technology that significantly reduced the experimental time. Despite the genus containing many pathogenically important species, only a handful of TF operator sites have been experimentally characterized for Burkholderia to date. Our study provides a significant addition to this knowledge base and illustrates some general challenges of discovering operators on a large scale for prokaryotes.

In this paper the ability to develop a gold-core, silver-shell surface-enhanced Raman scattering (SERS) nanosensor that has both a targeting component as well as a sensing component is demonstrated. The nanosensor is capable of targeting through the FcεRI receptor-mediated endocytic pathway due to the adsorption of 2,4-ε-dinitrophenol-L-lysine (DNP) ligand on the nanoparticle surface. The nanosensor is also sensitive to pH changes in the endocytic vesicle within the pH range of 4.5–7.5 by 4-mercaptopyridine (4-MPy) adsorbed to the particle surface. The targeting and sensing moieties do not significantly interfere with each other's function. The sensing component of the nanosensor is calibrated with in vivo measurements of rat basophil leukemia (RBL-2H3) cells in standard buffer solutions using the ionophore nigericin, which serves to equilibrate the external [H+] concentration with that of the cell compartments. The targeting of the nanosensor is verified with a β-hexosaminidase assay. It is also demonstrated that the targeted nanosensor is capable of making accurate cellular pH measurements in RBL-2H3 cells, out to ninety minutes after targeted nanosensor addition. Whole-cell, time-lapse, hyperspectral image cubes demonstrating endocytic vesicular pH changes during FcεRI receptor mediated endocytosis are demonstrated.

Physicochemical properties of DNA, such as shape, affect protein-DNA recognition. However, the properties of DNA that are most relevant for predicting the binding sites of particular transcription factors (TFs) or classes of TFs have yet to be fully understood. Here, using a model that accurately captures the melting behavior and breathing dynamics (spontaneous local openings of the double helix) of double-stranded DNA, we simulated the dynamics of known binding sites of the TF and nucleoid-associated protein Fis in Escherichia coli. Our study involves simulations of breathing dynamics, analysis of large published in vitro and genomic datasets, and targeted experimental tests of our predictions. Our simulation results and available in vitro binding data indicate a strong correlation between DNA breathing dynamics and Fis binding. Indeed, we can define an average DNA breathing profile that is characteristic of Fis binding sites. This profile is significantly enriched among the identified in vivo E. coli Fis binding sites. To test our understanding of how Fis binding is influenced by DNA breathing dynamics, we designed base-pair substitutions, mismatch, and methylation modifications of DNA regions that are known to interact (or not interact) with Fis. The goal in each case was to make the local DNA breathing dynamics either closer to or farther from the breathing profile characteristic of a strong Fis binding site. For the modified DNA segments, we found that Fis-DNA binding, as assessed by gel-shift assay, changed in accordance with our expectations. We conclude that Fis binding is associated with DNA breathing dynamics, which in turn may be regulated by various nucleotide modifications., Author Summary Cellular transcription factors (TFs) are proteins that regulate gene expression, and thereby cellular activity and fate, by binding to specific DNA segments. The physicochemical determinants of protein-DNA binding specificity are not completely understood. Here, we report that the propensity of transient opening and re-closing of the double helix, resulting from thermal fluctuations, aka “DNA breathing” or “DNA bubbles,” can be associated with binding affinity in the case of Fis, a well-studied nucleoid-associated protein in Escherichia coli. We found that a particular breathing profile is characteristic of high-affinity Fis binding sites and that DNA fragments known to bind Fis in vivo are statistically enriched for this profile. Furthermore, we used simulations of DNA breathing dynamics to guide design of gel-shift experiments aimed at testing the idea that local breathing influences Fis binding. As a result, we show that via nucleotide modifications but without modifying nucleotides that directly contact Fis, we were able to transform a low-affinity Fis binding site into a high-affinity site and vice versa. The nucleotide modifications were designed only based on DNA breathing simulations. Our study suggests that strong Fis-DNA binding depends on DNA breathing - a novel physicochemical characteristic that could be used for prediction and rational design of TF binding sites.

Recently, the development of an IgE receptor (FcεRI)-targeted, pH-sensitive, surface-enhanced Raman spectroscopy (SERS) nanosensor has been demonstrated by Nowak-Lovato and Rector (Appl Spectrosc 63:387-395, 2009). The targeted nanosensor enables spatial and temporal pH measurements as internalized receptors progress through endosomal compartments in live cells. Trafficking of receptor-bound nanosensors was compared at physiological temperature (37 °C) versus room temperature (25 °C). As expected, we observed markedly slower progression of receptors through low-pH endocytic compartments at the lower temperature. We also demonstrate the utility of the nanosensors to measure directly changes in the pH of intracellular compartments after treatment with bafilomycin or amiloride. We report an increase in endosome compartment pH after treatment with bafilomycin, an H(+) ATPase pump inhibitor. Decreased endosomal luminal pH was measured in cells treated with amiloride, an inhibitor of Na(+)/H(+) exchange. The decrease in amiloride-treated cells was transient, followed by a recovery period of approximately 15-20 min to restore endosomal pH. These experiments demonstrate the novel application of Raman spectroscopy to monitor local pH environment in live cells with the use of targeted SERS nanosensors.

Background The clustering of genes in a pathway and the co-location of functionally related genes is widely recognized in prokaryotes. We used these characteristics to predict the metabolic involvement for a Transcriptional Regulator (TR) of unknown function, identified and confirmed its biological activity. Results A software tool that identifies the genes encoded within a defined genomic neighborhood for the subject TR and its homologs was developed. The output lists of genes in the genetic neighborhoods, their annotated functions, the reactants/products, and identifies the metabolic pathway in which the encoded-proteins function. When a set of TRs of known function was analyzed, we observed that their homologs frequently had conserved genomic neighborhoods that co-located the metabolically related genes regulated by the subject TR. We postulate that TR effectors are metabolites in the identified pathways; indeed the known effectors were present. We analyzed Bxe_B3018 from Burkholderia xenovorans, a TR of unknown function and predicted that this TR was related to the glycine, threonine and serine degradation. We tested the binding of metabolites in these pathways and for those that bound, their ability to modulate TR binding to its specific DNA operator sequence. Using rtPCR, we confirmed that methylglyoxal was an effector of Bxe_3018. Conclusion These studies provide the proof of concept and validation of a systematic approach to the discovery of the biological activity for proteins of unknown function, in this case a TR. Bxe_B3018 is a methylglyoxal responsive TR that controls the expression of an operon composed of a putative efflux system. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-1142) contains supplementary material, which is available to authorized users.