Azospirillum brasilense improves rice growth under salt stress by regulating the expression of key genes involved in salt stress response, abscisic acid signaling, and nutrient transport, among others.

Major food crops, such as rice and maize, display severe yield losses (30-50%) under salt stress. Furthermore, problems associated with soil salinity are anticipated to worsen due to climate change. Therefore, it is necessary to implement sustainable agricultural strategies, such as exploiting beneficial plant-microbe associations, for increased crop yields. Plants can develop associations with beneficial microbes, including arbuscular mycorrhiza and plant growth-promoting bacteria (PGPB). PGPB improve plant growth via multiple mechanisms, including protection against biotic and abiotic stresses. Azospirillum brasilense, one of the most studied PGPB, can mitigate salt stress in different crops. However, little is known about the molecular mechanisms by which A. brasilense mitigates salt stress. This study shows that total and root plant mass is improved in A. brasilense-inoculated rice plants compared to the uninoculated plants grown under high salt concentrations (100 mM and 200 mM NaCl). We observed this growth improvement at seven- and fourteen days post-treatment (dpt). Next, we used transcriptomic approaches and identified differentially expressed genes (DEGs) in rice roots when exposed to three treatments: 1) A. brasilense, 2) salt (200 mM NaCl), and 3) A. brasilense and salt (200 mM NaCl), at seven dpt. We identified 786 DEGs in the A. brasilense-treated plants, 4061 DEGs in the salt-stressed plants, and 1387 DEGs in the salt-stressed A. brasilense-treated plants. In the A. brasilense-treated plants, we identified DEGs involved in defense, hormone, and nutrient transport, among others. In the salt-stressed plants, we identified DEGs involved in abscisic acid and jasmonic acid signaling, antioxidant enzymes, sodium and potassium transport, and calcium signaling, among others. In the salt-stressed A. brasilense-treated plants, we identified some genes involved in salt stress response and tolerance (e.g., abscisic acid and jasmonic acid signaling, antioxidant enzymes, calcium signaling), and sodium and potassium transport differentially expressed, among others. We also identified some A. brasilense-specific plant DEGs, such as nitrate transporters and defense genes. Furthermore, our results suggest genes involved in auxin and ethylene signaling are likely to play an important role during these interactions. Overall, our transcriptomic data indicate that A. brasilense improves rice growth under salt stress by regulating the expression of key genes involved in defense and stress response, abscisic acid and jasmonic acid signaling, and ion and nutrient transport, among others. Our findings will provide essential insights into salt stress mitigation in rice by A. brasilense.

Evolving Populations in Biofilms Contain More Persistent Plasmids.

Bacterial plasmids substantially contribute to the rapid spread of antibiotic resistance, which is a crisis in healthcare today. Coevolution of plasmids and their hosts promotes this spread of resistance by ameliorating the cost of plasmid carriage. However, our knowledge of plasmid-bacteria coevolution is solely based on studies done in well-mixed liquid cultures, even though biofilms represent the main way of bacterial life on Earth and are responsible for most infections. The spatial structure and the heterogeneity provided by biofilms are known to lead to increased genetic diversity as compared with well-mixed liquids. Therefore, we expect that growth in this complex environment could affect the evolutionary trajectories of plasmid-host dyads. We experimentally evolved Shewanella oneidensis MR-1 with plasmid pBP136Gm in biofilms and chemostats and sequenced the genomes of clones and populations. Biofilm populations not only maintained a higher diversity of mutations than chemostat populations but contained a few clones with markedly more persistent plasmids that evolved via multiple distinct trajectories. These included the acquisition of a putative toxin-antitoxin transposon by the plasmid and chromosomal mutations. Some of these genetic changes resulted in loss of plasmid transferability or decrease in plasmid cost. Growth in chemostats led to a higher proportion of variants with decreased plasmid persistence, a phenomenon not detected in biofilms. We suggest that the presence of more stable plasmid-host dyads in biofilms reflects higher genetic diversity and possibly unknown selection pressures. Overall, this study underscores the importance of the mode of growth in the evolution of antibiotic-resistant bacteria.

Rapid Discovery of Functional Small Molecule Ligands against Proteomic Targets through Library-Against-Library Screening.

Identifying "druggable" targets and their corresponding therapeutic agents are two fundamental challenges in drug discovery research. The one-bead-one-compound (OBOC) combinatorial library method has been developed to discover peptides or small molecules that bind to a specific target protein or elicit a specific cellular response. The phage display cDNA expression proteome library method has been employed to identify target proteins that interact with specific compounds. Here, we combined these two high-throughput approaches, efficiently interrogated approximately 10(13) possible molecular interactions, and identified 91 small molecule compound beads that interacted strongly with the phage library. Of 19 compounds resynthesized, 4 were cytotoxic against cancer cells; one of these compounds was found to interact with EIF5B and inhibit protein translation. As more binding pairs are confirmed and evaluated, the "library-against-library" screening approach and the resulting small molecule-protein domain interaction database may serve as a valuable tool for basic research and drug development.

Targeting multiple chorismate-utilizing enzymes with a single inhibitor: validation of a three-stage design.

Chorismate-utilizing enzymes are attractive antimicrobial drug targets due to their absence in humans and their central role in bacterial survival and virulence. The structural and mechanistic homology of a group of these inspired the goal of discovering inhibitors that target multiple enzymes. Previously, we discovered seven inhibitors of 4-amino-4-deoxychorismate synthase (ADCS) in an on-bead, fluorescent-based screen of a 2304-member one-bead-one-compound combinatorial library. The inhibitors comprise PAYLOAD and COMBI stages, which interact with active site and surface residues, respectively, and are linked by a SPACER stage. These seven compounds, and six derivatives thereof, also inhibit two other enzymes in this family, isochorismate synthase (IS) and anthranilate synthase (AS). The best binding compound inhibits ADCS, IS, and AS with K(i) values of 720, 56, and 80 microM, respectively. Inhibitors with varying SPACER lengths show the original choice of lysine to be optimal. Lastly, inhibition data confirm the PAYLOAD stage directs the inhibitors to the ADCS active site.

3-nitro-tyrosine as an internal quencher of autofluorescence enhances the compatibility of fluorescence based screening of OBOC combinatorial libraries.

In the one-bead-one-compound (OBOC) combinatorial method, compounds are constructed on bead resin via split-mix library synthesis such that multiple copies of the same compound are displayed on each bead. These libraries are rapidly screened with enzyme-linked colorimetric, fluorescent, radiometric, or whole-cell binding assays. While fluorescence-based probes are powerful tools in OBOC screening, their utility is greatly limited by the intrinsic fluorescence of many commonly used solid supports, (e.g. TentaGel), residual coupling reagents, and library compounds. To overcome this problem, we topologically partitioned TentaGel resin with a thin Fmoc-protected outer layer and an unprotected inner core. The inner core was derivatized with 3-nitro-tyrosine, followed by random peptide library construction. Spectral scans from a confocal microscope showed a dramatic decrease in the autofluorescence of blank beads and OBOC peptide libraries across a broad range of the optical spectrum. The quenching capacity of 3-nitro-tyrosine was also visualized in fluorescent micrographs. Using biotin/streptavidin as a model ligand/receptor system, we demonstrated a marked increase in visibility of three commercially available fluorescent probes binding to quenched beads, and increased feasibility of using a robust and efficient fluorescence-based, bead sorting platform known as COPAS. These data show that using 3-nitro-tyrosine as an internal quencher greatly enhances the compatibility of fluorescence-based applications and OBOC combinatorial screening.

Synthesis of flavonoid analogues as scaffolds for natural product-based combinatorial libraries.

The design and synthesis of flavonoid analogues as combinatorial scaffolds is reported. Using commercially available materials, we synthesized chalcones with fluoro and carboxy groups. Nitration of these compounds generated highly functionalized flavonoid scaffolds with an o-fluoronitrobenzene template. Subsequent cyclizations of these chalcones resulted in the formation of several flavone and flavonone scaffolds. One of the flavonones was chosen as the scaffold to synthesize flavonoid derivatives on the solid phase. A series of flavonoid derivatives were obtained in high yields, which demonstrates that these highly functionalized scaffolds can be used in the synthesis of natural product-based combinatorial libraries for drug discovery.

A spiroisoxazolinoproline-based amino acid scaffold for solid phase and one-bead-one-compound library synthesis.

An efficient, multigram synthesis of a spiroisoxazolinoproline-based amino acid, 7, requiring minimal purification, delivering good cis:trans diastereoselectivity (approximately 1:4), and providing good yields is reported. Surface-bound studies of the reduction of an arylnitro group in the presence of an isoxazoline ring with tin(II) dichloride dihydrate were undertaken to confirm the stability of the isoxazoline ring. Full derivitization of this spiroisoxazolinoproline-based amino acid scaffold was performed during the synthesis of a sample library with high yields and high purity that validated the efficiency of the chemistry that was employed in resin-bound library synthesis. A 129,600 member one-bead-one-compound (OBOC) library based on the scaffold 7 was synthesized utilizing a dual amino acid encoding method and bifunctionalization of TentaGel resin.

Aminodeoxychorismate synthase inhibitors from one-bead one-compound combinatorial libraries: "staged" inhibitor design.

4-Amino-4-deoxychorismate synthase (ADCS) catalyzes the first step in the conversion of chorismate into p-aminobenzoate, which is incorporated into folic acid. We aim to discover compounds that inhibit ADCS and serve as leads for a new class of antimicrobial compounds. This report presents (1) synthesis of a mass-tag encoded library based on a "staged" design, (2) massively parallel fluorescence-based on-bead screening, (3) rapid structural identification of hits, and (4) full kinetic analysis of ADCS. All inhibitors are competitive against chorismate and Mg(2+). The most potent ADCS inhibitor identified has a K(i) of 360 microM. We show that the combinatorial diversity elements add substantial binding affinity by interacting with residues outside of but proximal to the active site. The methods presented here constitute a paradigm for inhibitor discovery through active site targeting, enabled by rapid library synthesis, facile massively parallel screening, and straightforward hit identification.

On-bead combinatorial techniques for the identification of selective aldose reductase inhibitors.

Aldose reductase (AKR1B1; ALR2; E.C. 1.1.1.21) is an NADPH-dependent carbonyl reductase which has long been associated with complications resulting from the elevated blood glucose often found in diabetics. The development of effective inhibitors has been plagued by lack of specificity which has led to side effects in clinical trials. To address this problem, a library of bead-immobilized compounds was screened against fluorescently labeled aldose reductase in the presence of fluorescently labeled aldehyde reductase, a non-target enzyme, to identify compounds which were aldose reductase specific. Picked beads were decoded via novel bifunctional bead mass spec-based techniques and kinetic analysis of the ten inhibitors which were identified using this protocol yielded IC50 values in the micromolar range. Most importantly, all of these compounds showed a preference for aldose reductase with selectivities as high as approximately 7500-fold. The most potent of these exhibited uncompetitive inhibition versus the carbonyl-containing substrate D/L-glyceraldehyde with a Ki of 1.16 microM.

Slow-binding human serine racemase inhibitors from high-throughput screening of combinatorial libraries.

One-bead one-compound combinatorial chemistry together with a high-throughput screen based on fluorescently labeled enzyme allowed the identification of slow binding inhibitors of human serine racemase (hSR). A peptide library of topographically segregated encoded resin beads was synthesized, and several hSR-binding compounds were isolated, identified, and resynthesized for further kinetic study. Of these, several showed inhibitory effects with moderate potency (high micromolar K(I)s) toward hSR. A clear structural motif was identified consisting of 3-phenylpropionic acid and histidine moieties. Importantly, the inhibitors identified showed no structural similarities to the natural substrate, L-serine. Detailed kinetic analyses of the properties of selected inhibitors show that the screening protocol used here selectively identifies slow binding inhibitors. They provide a pharmacophore for the future isolation of more potent ligands that may prove useful in probing and understanding the biological role of hSR.

Discovery of selective aldo-keto reductase ligands--an on-bead assay strategy.

An enzyme labeling and screening strategy for the discovery of ligands selective in binding two structurally similar members of the aldo-keto reductase family of enzymes is reported. The resulting fluorescence microscope data obtained by screening a 74,088 member library led to the identification of selective ligands for aldose reductase (ALR2) and aldehyde reductase (ALR1). Resynthesis results validate the selectivity of these ligands.