Hybrid Two-Component Sensors for Identification of Bacterial Chemoreceptor Function.

Soil bacteria adapt to diverse and rapidly changing environmental conditions by sensing and responding to environmental cues using a variety of sensory systems. Two-component systems are a widespread type of signal transduction system present in all three domains of life and typically are comprised of a sensor kinase and a response regulator. Many two-component systems function by regulating gene expression in response to environmental stimuli. The bacterial chemotaxis system is a modified two-component system with additional protein components and a response that, rather than regulating gene expression, involves behavioral adaptation and results in net movement toward or away from a chemical stimulus. Soil bacteria generally have 20 to 40 or more chemoreceptors encoded in their genomes. To simplify the identification of chemoeffectors (ligands) sensed by bacterial chemoreceptors, we constructed hybrid sensor proteins by fusing the sensor domains of Pseudomonas putida chemoreceptors to the signaling domains of the Escherichia coli NarX/NarQ nitrate sensors. Responses to potential attractants were monitored by ß-galactosidase assays using an E. coli reporter strain in which the nitrate-responsive narG promoter was fused to lacZ Hybrid receptors constructed from PcaY, McfR, and NahY, which are chemoreceptors for aromatic acids, tricarboxylic acid cycle intermediates, and naphthalene, respectively, were sensitive and specific for detecting known attractants, and the ß-galactosidase activities measured in E. coli correlated well with results of chemotaxis assays in the native P. putida strain. In addition, a screen of the hybrid receptors successfully identified new ligands for chemoreceptor proteins and resulted in the identification of six receptors that detect propionate.IMPORTANCE Relatively few of the thousands of chemoreceptors encoded in bacterial genomes have been functionally characterized. More importantly, although methyl-accepting chemotaxis proteins, the major type of chemoreceptors present in bacteria, are easily identified bioinformatically, it is not currently possible to predict what chemicals will bind to a particular chemoreceptor. Chemotaxis is known to play roles in biodegradation as well as in host-pathogen and host-symbiont interactions, but many studies are currently limited by the inability to identify relevant chemoreceptor ligands. The use of hybrid receptors and this simple E. coli reporter system allowed rapid and sensitive screening for potential chemoeffectors. The fusion site chosen for this study resulted in a high percentage of functional hybrids, indicating that it could be used to broadly test chemoreceptor responses from phylogenetically diverse samples. Considering the wide range of chemical attractants detected by soil bacteria, hybrid receptors may also be useful as sensitive biosensors.

Mathematical and Structural Characterization of Strong Nonadditive Structure-Activity Relationship Caused by Protein Conformational Changes.

In medicinal chemistry, accurate prediction of additivity-based structure-activity relationship (SAR) analysis rests on three assumptions: (1) a consistent binding pose of the central scaffold, (2) no interaction between the R group substituents, and (3) a relatively rigid binding pocket in which the R group substituents act independently. Previously, examples of nonadditive SAR have been documented in systems that deviate from the first two assumptions. Local protein structural change upon ligand binding, through induced fit or conformational selection, although a well-known phenomenon that invalidates the third assumption, has not been linked to nonadditive SAR conclusively. Here, for the first time, we present clear structural evidence that the formation of a hydrophobic pocket upon ligand binding in PDE2 catalytic site reduces the size of another distinct subpocket and contributes to strong nonadditive SAR between two otherwise distant R groups.

Cooperative CO2 Absorption Isotherms from a Bifunctional Guanidine and Bifunctional Alcohol.

Designing new liquids for CO2 absorption is a challenge in CO2 removal. Here, achieving low regeneration energies while keeping high selectivity and large capacity are current challenges. Recent cooperative metal-organic frameworks have shown the potential to address many of these challenges. However, many absorbent systems and designs rely on liquid capture agents. We present herein a liquid absorption system which exhibits cooperative CO2 absorption isotherms. Upon introduction, CO2 uptake is initially suppressed, followed by an abrupt increase in absorption. The liquid consists of a bifunctional guanidine and bifunctional alcohol, which, when dissolved in bis(2-methoxyethyl) ether, forms a secondary viscous phase within seconds in response to increases in CO2. The precipitation of this second viscous phase drives CO2 absorption from the gas phase. The isotherm of the bifunctional system differs starkly from the analogous monofunctional system, which exhibits limited CO2 uptake across the same pressure range. In our system, CO2 absorption is strongly solvent dependent. In DMSO, both systems exhibit hyperbolic isotherms and no precipitation occurs. Subsequent 1H NMR experiments confirmed the formation of distinct alkylcarbonate species having either one or two molecules of CO2 bound. The solvent and structure relationships derived from these results can be used to tailor new liquid absorption systems to the conditions of a given CO2 separation process.

Selection for growth on 3-nitrotoluene by 2-nitrotoluene-utilizing Acidovorax sp. strain JS42 identifies nitroarene dioxygenases with altered specificities.

Acidovorax sp. strain JS42 uses 2-nitrotoluene as a sole source of carbon and energy. The first enzyme of the degradation pathway, 2-nitrotoluene 2,3-dioxygenase, adds both atoms of molecular oxygen to 2-nitrotoluene, forming nitrite and 3-methylcatechol. All three mononitrotoluene isomers serve as substrates for 2-nitrotoluene dioxygenase, but strain JS42 is unable to grow on 3- or 4-nitrotoluene. Using both long- and short-term selections, we obtained spontaneous mutants of strain JS42 that grew on 3-nitrotoluene. All of the strains obtained by short-term selection had mutations in the gene encoding the α subunit of 2-nitrotoluene dioxygenase that changed isoleucine 204 at the active site to valine. Those strains obtained by long-term selections had mutations that changed the same residue to valine, alanine, or threonine or changed the alanine at position 405, which is just outside the active site, to glycine. All of these changes altered the regiospecificity of the enzymes with 3-nitrotoluene such that 4-methylcatechol was the primary product rather than 3-methylcatechol. Kinetic analyses indicated that the evolved enzymes had enhanced affinities for 3-nitrotoluene and were more catalytically efficient with 3-nitrotoluene than the wild-type enzyme. In contrast, the corresponding amino acid substitutions in the closely related enzyme nitrobenzene 1,2-dioxygenase were detrimental to enzyme activity. When cloned genes encoding the evolved dioxygenases were introduced into a JS42 mutant lacking a functional dioxygenase, the strains acquired the ability to grow on 3-nitrotoluene but with significantly longer doubling times than the evolved strains, suggesting that additional beneficial mutations occurred elsewhere in the genome.

Three-dimensional conformal coatings through the entrapment of polymer membrane precursors.

We report a technique to coat polymers onto 3D surfaces distinct from traditional spray, spin, or dip coating. In our technique, the surface of a template structure composed of poly(lactic acid) swells and entraps a soluble polymer precursor. Once entrapped, the precursor is cured, resulting in a thin, conformal membrane. The thickness of each coating depends on the coating solution composition, residence time, and template size. Thicknesses ranged from 400 nm to 4 µm within the experimental conditions we explored. The coating method was compatible with a range of polymers. Complicated 3D structures and microstructures of 10 µm thickness and separation were coated using this technique. The templates can also be selectively removed, leaving behind a hollow membrane structure in the shape of the original printed, extruded, or microporous template structures. This technique may be useful in applications that benefit from three-dimensional membrane topologies, including catalysis, separations, and potentially tissue engineering.