It is well known boronic acids have its unique chemistry and related applications in organic synthesis. The boronic acid functionally group also plays very important roles in medicinal chemistry and chemical biology. For example, boronic acids have been developed as potential therapeutic agents, chemical biology tools. All these applications are directly related to the unique electronic and chemical properties of the boronic acid group. Herein, several application of boronic acids have been studied: 1) several groups of compounds were found as bacterial quorum sensing inhibitors; 2) a boronate compound was developed as a probe for detecting reactive oxygen species (ROS); and 3) boronic acid-modified aptamers can be used for glycoprotein recognition.

Carbohydrates are known to play important roles in a large number of physiological and pathological processes. Conceivably, “binders” of carbohydrates of biological importance could be used as diagnostic and therapeutic agents. Currently, lectins are the major available tools in research for carbohydrate recognition. However, the available lectins often have cross-reactivity issues, along with the high costs and stability issues. Therefore, there is a critical need to develop alternatives (lectin mimics). In this regard, there have been very active efforts in developing different “binders”, such as small molecule lectinmimics and aptamers. Among all the small molecule lectinbmimics developments, boronic acid stands out as the most important building blocks of the sensors design for carbohydrates biomarkers due to its intrinsic binding affinities with diols. To address a fundamental question that whether boronic acid also binds to six-membered ring sugars, with very limited precedents, we provided a concrete experimental evidence of the binding. Specifically, a series of isoquinolinylboronic acids were found to have remarkably high binding affinities with fluorescence change upon binding to representative sugars. Most importantly, these isoquinolinylboronic aicds showed weak but very encouraging bindings with six-membered sugar model. All these promising results paves the way of using boronic acids, especially isoquinolinylboronic acid as building blocks for chemosensors design for biological carbohydrates biomarkers, which universally contain six-membered ring and liner diols. Aptamer provides another alternative way for sensors development for carbohydrates biomarkers as lectin mimics. Compared to lectins, they are normally cheaper and more stable. However, there is much less options. Another challenging area for aptamer-based lectin mimics development is the difficulty to differentiate changes in glycosylation patterns of a glycoprotein, which affect the function of a glycoprotein and thus recognized as biomarkers. To address this major challenge, our group first demonstrated that the incorporation of a boronic acid into DNA would allow for the aptamer selection process to gravitate towards the glycosylation site. To examine the generality of boronic acid incorporation, increase the structural diversity, and broaden the application of boronic acid-modified DNA, a series of B-TTP analogues with simplified structures were designed, synthesized, and successfully incorporated into DNA. A simple route was also developed using 1,7-octadiyne as a linker for both Sonogashira coupling with thymidine and CuAAC tethering of a boronic acid moiety. This paves the way for the preparation of a large number of B-TTPs with different structural features for aptamer selection or array analysis. Finally, bacterial quorum sensing has received much attention in recent years because of its relevance to pathological events such as biofilm formation. As one of the very first groups that developed a series of antagonists for AI-2 mediated quorum sensing, we herein designed and synthesized a series of analogues based on the structures of two lead inhibitors identified through virtual screening. Besides, we also examined their inhibitory activities, twelve of which showed equal or better inhibitory activities compared with the lead inhibitors. The best compound showed an IC50 of about 6 mM in a whole cell assay using Vibrio harveyi as the model organism. This encouraging results and SAR discuss also paves the way for the finding of more potent compound through further structure optimization.

The members of the genus Bacillus produce a wide variety of secondary metabolites with antimetabolic and pharmacological activities. These metabolites are mostly small peptides and have unusual components and chemical bonds. These metabolites are synthesized nonribosomally by multifunctional enzyme complexes called peptide synthetases. One of those small peptides, bacilysin, is a dipeptide antibiotic composed of L-alanine and L-anticapsin which is produced and excreted by certain strains of Bacillus subtilis. Proteins that are responsible to synthesize bacilysin are encoded by bac operon. It has been shown that the biosynthesis of bacilysin is under the control of quorum sensing global regulatory pathway through the action of ComQ/ComX, PhrC (CSF), ComP/ComA in a Spo0K (Opp)-dependent manner. The objective of the study is to identify the functional roles of bacilysin biosynthesis in the regulatory cascade and idiophase cell physiology operating in B. subtilis by using gel-based and gel-free proteomics techniques. For this, we employed comparative proteome-wide analysis of the bacilysin producer B. subtilis PY79 and its bacilysin nonproducer derivative bacA::lacz::erm OGU1 strain which was recently constructed by our group. Identification via GeLC analysis of 76 differentially expressed proteins from total soluble proteome of wild-type PY79 and bacilysin minus OGU1 strain indicated the direct or indirect multiple effects of bacilysin on metabolic pathways, global regulatory systems and sporulation.

This thesis focuses on the analysis of the role of adhesion between substrate and cell and factors of Quorum sensing on the migration of Dictyostelium amoeba. Tools to automate the recordings of videomicroscopy and image analysis have been developed to work with very large samples of cells and toquantify cell migration. A microfluidic device for cell detachment in hydrodynamic flow combined witha motorized stage has allowed a statistical study of adhesion but also the dynamics of detachment. The analysis of the migration of Dictyostelium in non nutritive medium highlights the role of density on celldifferentiation and migration capacity. We observe the presence of a maximum speed of migration after6 hours of starvation. We show that the adhesion to glass is twice as low in deprivation buffer as inthe nutrient medium. The experiences of migration in growth medium revealed the presence of a factorof detection of density secreted by the cells and regulating their random migration. The diffusion coefficient, the persistence of the movement and morphology of cells vary depending on the concentrationof this factor. This factor does not affect cell adhesion but only the dynamics of detachment. Finally, the testing protocol developed allowed us to make a comparative study of migration by varying otherparameters such as surface or the chemical composition of experimental medium. This work concludesby outlining the possible role of adhesion to the migration of Dictyostelium in nutrient medium.

Quorum sensing (QS) cell-cell communication systems are utilized by bacteria to coordinate their behaviour according to cell density. Several different types of QS signal molecules have been identified, among which acyl-homoserine lactones (AHLs) produced by Proteobacteria have been studied to the greatest extent. QS has been shown to be involved in many aspects of bacterial life, including virulence, bioluminescence, symbiosis, antibiotic production, swarming and swimming motility, biofilm formation, conjugation and growth inhibition. Although QS has been studied extensively in cultured microorganisms, little is known about the QS systems of uncultured microorganisms and the roles of these systems in microbial communities. To extend our knowledge of QS systems and to better understand the signalling that takes place in the natural environment, in the first part of this thesis, isolation and characterization of new QS systems from metagenomic libraries constructed using DNA from activated sludge and soil were described. Using an Agrobacterium biosensor strain, three cosmids (QS6-1, QS10-1 and QS10-2) that encode the production of QS signals were identified and DNA sequence analysis revealed that all three clones encode a novel luxI family AHL synthase and a luxR family transcriptional regulator. Thin layer chromatography revealed that these LuxI homolog proteins are able to synthesize multiple AHL signals. Tandem mass spectrometry analysis revealed that LuxIQS6-1 directs the synthesis of at least three AHLs, 3-O-C14:1 HSL, 3-O-C16:1 HSL and 3-O-C14 HSL; LuxIQS10-1 directs the synthesis of at least 3-O-C12 HSL and 3-O-C14 HSL; while LuxIQS10-2 directs the synthesis of at least C8 HSL and C10 HSL. Two possible new AHLs, C14:3 HSL and (?)-hydroxymethyl-3-O-C14 HSL, were also found to be synthesized by LuxIQS6-1. Agrobacterium tumefaciens is a plant pathogen that causes crown gall disease. Its ability to transfer and integrate foreign DNA into plant genome also makes it a useful tool for plant genetic engineering. Ethylene, the gaseous plant hormone, has been reported to be important for both crown gall development and A. tumefaciens mediated transformation efficiency to plants. ACC deaminase, an enzyme that can break down ACC, the direct precursor of ethylene biosynthesis in plants, is a mechanism used by some plant growth promoting bacteria (PGPB) to promote plant growth by reducing stress ethylene levels. In the second part of this thesis, the effect of ACC deaminase on A. tumefaciens induced crown gall development and on A. tumefaciens mediated transformation efficiency was studied. By either introduction of an ACC deaminase encoding gene into the virulent strain A. tumefaciens C58 or co-inoculation of A. tumefaciens C58 with an ACC deaminase containing PGPB P. putida UW4, using different plant systems including tomato plants and castor bean plants, it was found that the presence of an ACC deaminase significantly inhibited crown gall development. It was also found that introduction of an acdS gene into the disarmed A. tumefaciens strain GV3101::pMP90 reduced the ethylene levels evolved by plants during infection and cocultivation process and increased the transformation efficiency of commercialized canola cultivars. The A. tumefaciens D3 strain was reported to contain an ACC deaminase encoding gene (acdS). In this study it was determined that this strain is an avirulent strain and shows plant growth promoting activity. When co-inoculated with A. tumefaciens C58 on castor bean stems, both the wild type and the acdS knockout mutant showed biocontrol activity and were able to significantly inhibit crown gall formation, with the wild type strain showing slightly better tumor inhibition effects. The mutation of acdS and its regulatory gene lrpL in A. tumefaciens D3 was also found to affect QS signal production of this strain, which indicates a cross talk between the two sets of genes.

Bacterial quorum sensing is regarded as a novel target for the design of antimicrobials. Based on lead structures identified from HTS, 39 analogues have been synthesized and evaluated in Vibrio haveyi. Potent inhibitors with IC50 values at single-digit micromolar concentrations for AI-2 mediated quorum sensing have been identified. On the second project, post-synthesis modifications of DNA provide easy functionalizations for expanded applications such as aptamer selection. A CBT-modified thymidine analogue (CBT-TTP) has been synthesized and used for enzymatic incorporation into DNA. Post-synthesis modifications through condensation with 1,2-aminothiol for installation of a boronic acid moiety or a fluorophore have been achieved. On the third project, H2S has been recognized as an important gasotransmitter and its concentration is relevant to a variety of diseases. A novel fluorescent probe (DNS-Az) has been developed for quantitation of H2S in aqueous solutions. This probe has been used to measure H2S concentrations in the blood.

The bacterial pathogen Vibrio cholerae is responsible for ongoing cholera outbreaks in Haiti and elsewhere. Association of V. cholerae with the human host is responsible for fatal disease, but the bacteria also reside as natural inhabitants of aquatic environments, commonly attaching as biofilms to chitinous surfaces of copepods and crabs. Prior studies in V. cholerae demonstrated that competence for genetic transformation, a mechanism of horizontal gene transfer (HGT), requires the TfoX regulator protein that is triggered by chitin, and the HapR transcription factor that is made in response to quorum sensing (QS) signals produced by V. cholerae and Vibrios. To define regulatory components connecting extracellular signals to natural competence, I first demonstrated that QS molecules produced by Vibrios within multi-species chitinous biofilms are required for DNA uptake by V. cholerae, confirming the critical role of QS signals in HGT. Second, I identified by transposon-mutagenesis a new positive regulator of competence, CytR (cytidine repressor), only studied prior in E. coli as a regulator of nucleoside scavenging. Specific mutations in V. cholerae CytR impaired expression of competence genes and halted DNA uptake; and the addition of exogenous cytidine had similar affects as predicted in E. coli. V. cholerae and other competent Vibrios encode TfoX, HapR, and CytR, although none of these regulators directly controls genes coding for the DNA uptake apparatus. Thus, these results have uncovered a regulatory network, likely used by many Vibrios, that contains additional factors linking several extracellular chemical molecules (cytidine, chitin, and QS signals) to DNA uptake. My study has begun to define a molecular mechanism by which both environment and genetics contribute to genome evolution for this important marine pathogen.

Quorum sensing (QS) cell-cell communication systems are utilized by bacteria to coordinate their behaviour according to cell density. Several different types of QS signal molecules have been identified, among which acyl-homoserine lactones (AHLs) produced by Proteobacteria have been studied to the greatest extent. QS has been shown to be involved in many aspects of bacterial life, including virulence, bioluminescence, symbiosis, antibiotic production, swarming and swimming motility, biofilm formation, conjugation and growth inhibition. Although QS has been studied extensively in cultured microorganisms, little is known about the QS systems of uncultured microorganisms and the roles of these systems in microbial communities. To extend our knowledge of QS systems and to better understand the signalling that takes place in the natural environment, in the first part of this thesis, isolation and characterization of new QS systems from metagenomic libraries constructed using DNA from activated sludge and soil were described. Using an Agrobacterium biosensor strain, three cosmids (QS6-1, QS10-1 and QS10-2) that encode the production of QS signals were identified and DNA sequence analysis revealed that all three clones encode a novel luxI family AHL synthase and a luxR family transcriptional regulator. Thin layer chromatography revealed that these LuxI homolog proteins are able to synthesize multiple AHL signals. Tandem mass spectrometry analysis revealed that LuxIQS6-1 directs the synthesis of at least three AHLs, 3-O-C14:1 HSL, 3-O-C16:1 HSL and 3-O-C14 HSL; LuxIQS10-1 directs the synthesis of at least 3-O-C12 HSL and 3-O-C14 HSL; while LuxIQS10-2 directs the synthesis of at least C8 HSL and C10 HSL. Two possible new AHLs, C14:3 HSL and (?)-hydroxymethyl-3-O-C14 HSL, were also found to be synthesized by LuxIQS6-1. Agrobacterium tumefaciens is a plant pathogen that causes crown gall disease. Its ability to transfer and integrate foreign DNA into plant genome also makes it a useful tool for plant genetic engineering. Ethylene, the gaseous plant hormone, has been reported to be important for both crown gall development and A. tumefaciens mediated transformation efficiency to plants. ACC deaminase, an enzyme that can break down ACC, the direct precursor of ethylene biosynthesis in plants, is a mechanism used by some plant growth promoting bacteria (PGPB) to promote plant growth by reducing stress ethylene levels. In the second part of this thesis, the effect of ACC deaminase on A. tumefaciens induced crown gall development and on A. tumefaciens mediated transformation efficiency was studied. By either introduction of an ACC deaminase encoding gene into the virulent strain A. tumefaciens C58 or co-inoculation of A. tumefaciens C58 with an ACC deaminase containing PGPB P. putida UW4, using different plant systems including tomato plants and castor bean plants, it was found that the presence of an ACC deaminase significantly inhibited crown gall development. It was also found that introduction of an acdS gene into the disarmed A. tumefaciens strain GV3101::pMP90 reduced the ethylene levels evolved by plants during infection and cocultivation process and increased the transformation efficiency of commercialized canola cultivars. The A. tumefaciens D3 strain was reported to contain an ACC deaminase encoding gene (acdS). In this study it was determined that this strain is an avirulent strain and shows plant growth promoting activity. When co-inoculated with A. tumefaciens C58 on castor bean stems, both the wild type and the acdS knockout mutant showed biocontrol activity and were able to significantly inhibit crown gall formation, with the wild type strain showing slightly better tumor inhibition effects. The mutation of acdS and its regulatory gene lrpL in A. tumefaciens D3 was also found to affect QS signal production of this strain, which indicates a cross talk between the two sets of genes.

In Bacillus subtilis, genetic competence, sporulation and antibiotic production are controlled by quorum-sensing global regulatory mechanism. Bacilysin, being produced and excreted by certain strains of Bacillus subtilis, is a dipeptide antibiotic composed of L-alanine and L-anticapsin. We showed that the biosynthesis of bacilysin is under the control of quorum sensing global regulatory pathway through the action of ComQ/ComX, PhrC (CSF), ComP/ComA in a Spo0K (Opp)-dependent manner. Recently, the ywfBCDEF genes of B. subtilis 168 were shown to carry biosynthetic core function and renamed as bacABCDE operon. The objective of the present study is to elucidate the effects of previously-identified genes srfA, oppA, comA, phrC, phrF, phrK, comQ (comX), comP, spo0H, spo0A, abrB and codY on the expression of bacilysin biosynthetic operon bacABCDE. In order to monitor the expression of bac operon a B. subtilis strain, namely OGU1, containing a transcriptional bacA-lacZ fusion at bacA locus was constructed. Subsequently, each of the above-mentioned genes of cell density signaling was insertionally inactivated by transforming the competent cells of OGU1 with chromosomal DNA of the corresponding blocked mutant strains. The resulting strains and OGU1 as the control were cultured in PA medium and bacA-directed &amp / #946 / -galactosidase activities were monitored. bacA-lacZ expression was severely impaired in the srfA, oppA, comA, phrC, phrF, phrK, comQ (comX), comP, spo0H and spo0A disrupted mutants. On the other hand, in the abrB single mutant bacA expression level increased nearly 2-fold during exponential growth and in the codY mutant it severely decreased during the stationary phase.

The members of the genus Bacillus produce a wide variety of secondary metabolites with antimetabolic and pharmacological activities. Most of these metabolites are small peptides that have unusual components and chemical bonds and synthesized nonribosomally by multifunctional enzyme complexes called peptide synthetases. Bacilysin, being produced and excreted by certain strains of Bacillus subtilis, is one of the simplest peptide antibiotics known. It is a dipeptide with an N-terminal L-alanine and an unusual amino acid, L-anticapsin, at its C-terminal. Recently, ywfBCDEF operon of B. subtilis 168 was shown to carry bacilysin biosynthesis function, the genes of this operon were renamed as bacABCDE. The first member of bac operon, bacA gene was proved to encode the function of L-alanine &ndash / L-anticapsin amino acid ligation. Bacilysin production is regulated at different levels, negatively by GTP via the transcriptional regulator CodY and AbrB while positive regulation occurs by guanosine 5