Ncd is a member of the kinesin family of motor proteins. Ncd is involved in the processes of meiosis and early mitosis in <i>D. melanogaster</i>. PCR-mediated site-directed mutagenesis was utilized to introduce specific mutations into pET/MC6, a construct containing the motor domain of ncd. Six mutations were generated, two at glutamic acid residue 656, two at proline residue 649, one at arginine residue 623, and one double mutant at arginine residue 623 and threonine residue 632. Mutants proteins were expressed in bacteria and further characterized. Mutagenesis of the proline or glutamic acid residues resulted in insoluble proteins. The one exception is the mutagenesis of glutamic acid residue 656 into a glutamine, which resulted in a partially soluble protein. Mutagenesis of the arginine residue into an alanine (MC6-A623) resulted in a soluble protein while the double mutation of the arginine and threonine was insoluble. MC6-A623 exhibited a similar S-sepharose ion exchange chromatography binding and elution profile as MC6. Peptide antibodies made to conserved ncd motor domain sequences also recognized MC6- A623. The affinity of MC6-A623 (under the conditions tested) for microtubules was less than MC6. Most interestingly, under the conditions tested, MC6-A623 did not exhibit an increased ATPase rate in the presence of microtubules, a hallmark of the kinesin family of microtubule motor proteins. Analysis of the published ncd crystal structure, other motor protein sequences, and the experimental results of the mutagenesis of arginine residue 623, suggest that this residue is involved in the binding of MC6 to microtubules. / Master of Science

Bacterial fruit blotch (BFB) of cucurbits is caused by Acidovorax citrulli, a Gram-negative seedborne bacterium that can cause up to 100% fruit yield losses in the field. Currently, BFB is a major problem for the cucurbits industry worldwide. Thus far, attempts to identify resistance in cucurbit germplasm for controlling BFB have been unsuccessful. Despite the importance of the disease, little is known about the molecular mechanisms of A. citrulli pathogenicity, due to a lack of molecular tools for studying the A. citrulli/cucurbit interaction. The genomic sequence of A. citrulli strain AAC00-1 has been determined, and the components of type III secretion system have been identified. The goal of this research was to develop molecular tools for studying the BFB disease. Nineteen putative type III effector genes were cloned from two representative A. citrulli strains (AAC00-1 and M6). The distribution of 19 type III effectors among A. citrulli strains, collected worldwide, was studied. A novel Gateway-compatible binary vector was developed for transient expression of A. citrulli type III effectors genes in planta. A set of modified vectors for marker-exchange mutagenesis in A. citrulli were constructed. The model plant species Nicotiana benthamiana was found to be susceptible to A. citrulli, while Nicotiana tabacum was resistance to A. citrulli, so therefore could carry nonhost resistance genes. Two T3S effectors, Aave1548 and Aave2166, triggered water soaking-like cell death in N. benthamiana, but HR-like cell death in N. tabacum. Bacterial mutagenesis and in planta disease assay confirmed that both Aave1548 and Aave2166 have significant virulence contributions to A. citrulli in N. benthamiana plant and melon seeds. Aave2166 encodes a putative acetyltransferase that belongs to the YopJ super family, which is conserved in both animal and plant pathogenic bacteria. Wild type but not the putative catalytic mutant (C232A) of Aave2166 can trigger cell death phenotype in N. benthamiana and N. tabacum. N. benthamiana yeast two-hybrid cDNA library screening using Aave2166 identified six N. benthamiana proteins/peptides which specifically interacted with Aave2166. Further characterization of these Aave2166 interactors may allow us to understand the virulence mechanism provided by Aave2166. The identification of nonhost resistance genes that can recognize Aave2166 and other type III effectors may help to develop novel strategies to control BFB disease of cucurbit. / Ph. D.

DNA serves as a mode of storage for genetic information and carries essential biophysical functions which continue to gain importance for new biomolecular applications. The functionalization of proteins with nucleic acids has found several different uses in a variety of fields such as DNA origami due to the highly programmable nature of DNA. The modulation of key biomolecular properties of proteins by the conjugation of DNA nanotools, mechanical forces and potential catalytic modulators is an under discovered area of protein engineering. In this doctoral thesis work, functionalization strategies of proteins with DNA oligonucleotides, the catalytic mechanism of AdhD from Pyrococcus furiosus and effects of mechanical forces on enzymatic catalysis have been investigated through the utilization of DNA tools. In the first part of the thesis (Chapter 2), several bioconjugation strategies which can be used to attach DNA oligonucleotides to proteins have been reviewed and assessed for emerging biomolecular applications. This work provides insights about commonly used bioconjugation reactions and investigates the performance of each conjugation reaction in terms of yield, site- specificity, flexibility in conjugation position, steric hinderance, cost, reagent availability and risk of altering native protein properties. In the next section of the thesis (Chapter 3), DNA spring attachment sites are strategically modeled and created on the model enzyme for assigned bioconjugation strategies and the DNA springs are assembled on this model enzyme. The effect of the forces generated by the DNA springs during bulk catalysis is investigated and the changes in binding pockets and substrate specificity properties are demonstrated. A novel magnetic bead-based purification strategy for the separation of DNA spring conjugated enzyme is established in this work to ensure homogenous catalysis conditions. This construct allows the tuning of catalytic properties by the usage of different lengths of DNA and is shown to be reversible. As enzymatic catalysis is investigated under bulk conditions and the amino acid sequence within the active site is not altered, this strategy provides a new platform for the modulation of enzymatic trajectories without isolation and altered microenvironment limitations as well as the irreversible effects of conventional techniques such as mutagenesis and directed evolution. In the last part of the thesis (Chapter 4), the effects of different DNA constructs on AdhD are investigated. Convex DNA springs are constructed on AdhD molecules with the goal of applying compressive forces over the active site. In addition, DNA tweezers are designed to apply comparable forces to the DNA spring studied in Chapter 3, assembled on AdhD and purified using the same magnetic bead-based strategy. This construct, as discussed in Chapter 1, allows the modulation of substrate specificity profiles consistent with its mechanical design. As a continuation of the last part of the thesis, the rare earth element (REE) binding capacity of AdhD and the effect of metal binding on its enzymatic trajectory are investigated in Chapter 5. The rare earth element (REE) binding affinity of AdhD and its effect on enzymatic catalysis on both the forward and reverse reactions are demonstrated with and without the presence of a metal chelator. This discovery sheds light on the potential allosteric regulation mechanisms of AdhD and the catalytic regulator effect of REEs. The work presented in this doctoral thesis demonstrates different biomolecular approaches towards the modulation of enzymatic properties through DNA oligonucleotide conjugation, application of mechanical forces and potential catalytic regulators. In the future, the results presented in this work can be utilized to initiate in depth studies about protein-DNA conjugation and modulation of enzymatic catalysis, and discover extended applications which can be used universally across different biomolecular platforms.

Francisella tularensis is a Gram-negative, facultative intracellular coccobacillus and the etiologic agent of tularemia for a wide variety of vertebrate and invertebrate animal species. Several species and subspecies of Francisella are currently recognized. However, the majority of infections are caused by F. tularensis subspecies tularensis (type A) and subspecies holarctica (type B). Given the low infectious dose, multiple transmission routes, severity of illness, and lack of licensed vaccines, F. tularensis has long been considered a potential biological weapon and is now classified as a category A select agent by the National Institutes of Health and the Centers for Disease Control and Prevention. The investigation of the mechanisms of pathogenesis by F. tularensis type A and B strains is hindered by the difficulty and lack of methods to mutate the putative genes that encode for virulence factors. New genetic tools have been developed that have enabled mutagenesis of F. tularensis type A and type B stains. However, site-specific mutations remain difficult to execute or these methods generate random mutations. In this study a novel method was developed to create site-directed mutations in a putative capsule biosynthesis locus to knock out encapsulation of the attenuated F. tularensis live vaccine strain. Two suicide vectors for mutagenesis of F. tularensis were constructed based on the commercial PCR cloning vector pSC-A. These vectors were created by inserting into the cloning site a kanamycin resistance gene boarded upstream by 1.3 kb of N-terminal DNA and downstream by 1.3 kb of C-terminal DNA that flanks the target gene. Cryotransformation was used to introduce the vectors into F. tularensis. Open reading frame (ORF) FTT0793, which may encode for an ABC transporter involved in capsule export, was initially selected for mutagenesis in order to generate a mutant that was nonencapsulated, but could still synthesize capsule and induce a host immune response. Mutagenesis of this gene was successful. However, phenotypic assays could not confirm that the mutant was nonencapsulated compared to the parent. Therefore, adjacent ORFs FTT0798 and FTT0799, which may encode for a galactosyl transferase and mannosyl transferase, respectively, were also deleted to completely knock out capsule synthesis. The resulting mutant appeared to be nonencapsulated as determined by negative staining transmission electron microscopy. In this study, a plasmid and method for generating allelic exchange mutants is reported, which should be useful for generating additional mutants of F. tularensis for use in clarifing the roles of specific genes. This vector is currently being used to make a nonencapsulated mutant of a virulent type A strain to determine the role of capsule in virulence. / Master of Science

Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen that is a substantial threat, particularly in hospital settings, causing severe infections in immunocompromised patients that may lead to death. Pseudomonas aeruginosa harbors a multitude of virulence factors that enable this pathogen to establish both acute and chronic infections in humans. A key determinant of acute infections is a hollow molecular needle structure used for injecting toxins into a host cell, called the type three secretion system (T3SS). The secretion machinery itself is highly complex and, together with the specific secreted factors, requires expression of more than 30 genes. Due to the high energy cost of its synthesis to the organism this system is highly regulated to finely time gene expression to coincide with host contact. ExsA, a member of the AraC-type transcription factor family, is the main transcriptional activator of all the genes necessary for expression of the T3SS. Members of the AraC family are characterized by the presence of two helix-turn-helix (HTH) motifs, which bind to the promoter DNA and activate transcription. ExsA uses its HTH containing C-terminal domain (CTD) to regulate gene expression from 10 different promoters. The N-terminal domain (NTD) of ExsA mediates dimerization and regulation of ExsA-activity. While most AraC-type activators are regulated by a small molecule ligands, ExsA is regulated by another protein, ExsD. As part of a four-protein signaling cascade, ExsD interacts directly with ExsA to prevent transcription of T3SS-associated genes under non-inducing conditions prior to host cell contact. The entire regulatory cascade includes of two additional proteins, ExsC and ExsE. ExsA, ExsC, ExsD, and ExsE follow a partner-switching mechanism to link expression of the secretion system with host cell contact. Our laboratory is working to understand this unique signaling mechanism by determining the molecular basis for the regulation of this important virulence factor. Previous studies in the laboratory have solved the structures of ExsE, ExsC and ExsD, and shed light on how these proteins interact and compete for overlapping binding sites. However, it is still unclear as to how the ExsA and ExsD interact and thus how regulation is mediated at the molecular level. In the presented study, we sought to map the molecular interface between ExsA and ExsD. First, the crystal structure of ExsA-NTD is presented wherein the dimerization interface of the protein was identified. Two of the well-studied AraC-type proteins, AraC and ToxT crystal structures have been solved by others in the presence of their respective ligands. Residues that were involved in ligand binding in AraC and ToxT were aligned with the residues in ExsA and analyzed for interaction with ExsD. However, this canonical binding pocket appeared to be not involved in the interaction between ExsA and ExsD. Structure directed site-specific mutagenesis was carried out to construct many different variants of ExsD and ExsA. Thus constructed variants were purified and analyzed in a functional assay. Using this approach, we were able to identify regions on ExsD and ExsA that are crucial for the interaction and for the regulation of ExsA-dependent transcription. It turns out that backbone interactions between the amino-terminal residues of ExsD and the beta-barrel region of the ExsA-NTD are pivotal. This result explains how ExsA and ExsC compete for ExsD binding, since both target the same regions on ExsD. / PHD / Pseudomonas aeruginosa is an opportunistic pathogen that is notorious for causing severe infections in immunocompromised individuals. Acute Pseudomonas aeruginosa infections are characterized by immediate adverse effects. An initial acute infection may become chronic, leading to long-term morbidity and mortality in affected individuals. During the initial stages of infection P. aeruginosa uses the type three secretion system, a syringe-like structure, to puncture the host cell and inject potent toxins. The activation of the genes required for forming this structure is tightly controlled by an activator protein, ExsA. When P.aeruginosa is not invading a host, ExsA is inhibited by another protein called ExsD, to prevent the needless production of the secretion apparatus. The presented work explores the mechanism of how ExsD achieves this inhibition of ExsA. This information is of potential biomedical interest because a clear understanding of the molecular basis for the interaction could inform the development of a small-molecule mimic of ExsD to be used in therapy. In Chapter 2 we report the structure of the domain of ExsA that is known to bind ExsD. Also, in this chapter and more so in Chapter 3, we performed a detailed analysis of potential interacting regions and ultimately succeeded in identifying key interacting regions in both ExsA and ExsD.

O objetivo deste estudo foi aumentar o banco de dados em relação a resíduos (cake) e efluentes (licor) industriais e o seu nível de clastogenicidade. Este estudo contribuiu para mostrar: a) que o bioensaio com Tradescantia pallida foi sensível para a avaliação da clastogenicidade em mistura complexa de resíduos catalíticos industriais, nunca testados anteriormente. b) a tendência de uma dose resposta para ambos os resíduos catalíticos c)a pasta (cake) apresenta maior clastogenicidade que o licor nas concentrações estudadas. Provavelmente isto se deve a menor concentração de Ti e Al no licor do que no cake. / The aim of this study was to increase data concerning liquid effluent (liquor) and solid waste (cake) and their level of clastogenicity using TradMCN. This study contributed to show a) bioassay Trad-MCN with Tradescantia pallida was sensitive to evaluate the clastogenicity in a complex waste mixture, never tested before b) a tendency of a dose response for both catalytic wastes. c) higher clastogenicity of cake comparing to liquor effluent in concentrations evaluated. Probably this is due to the much lower Ti and Al concentrations in the liquor than in the cake

La transcétolase (TK, EC 2.2.1.1) est une enzyme catalysant la formation de cétoses de configuration D-thréo à partir d’aldéhydes α-hydroxylés (2R), par formation stéréospécifique d’une liaison C-C. L’objectif de ces travaux est d’inverser l’énantiosélectivité de cette enzyme par ingénierie afin d’obtenir des cétoses L-érytho (recherchés pour leurs applications potentielles dans les domaines pharmaceutique et/ou nutritionnel) à partir d’aldéhydes α-hydroxylés (2S). Dans ce but, une TK thermostable (mTKgst) issue de la bactérie thermophile Geobacillus stearothermophillus a d’abord été identifiée et produite. L’étude de sa structure tridimensionnelle a permis d’identifier deux résidus du site actif ayant un rôle potentiel dans l’inversion de son énantiosélectivité : Leu382 et Asp470. Des banques demTKgst mutées ont alors été créées, selon deux stratégies : rationnelle et semi-rationnelle. La première a consisté à muter les deux résidus sélectionnés par mutagenèse par saturation de site, tandis que la seconde a consisté à modifier deux séquences de cinq résidus contigus à aux positions clés, selon la mutagenèse par cassette. Afin d’identifier les mTKgst mutées d’intérêt, un test de criblage à haut-débit a été mis au point, basé sur le suivi pH-métrique de la réaction en présence de rouge de phénol. A l’issue du criblage, le variant mTKgst-L382D/D470S a été mis en évidence. Son activité vis-à-vis d’un aldéhyde modèle de configuration (2S) a été augmentée d’un facteur 5 par rapport à l’enzyme sauvage et la perte de l’énantiosélectivité vis-à-vis desaldéhydes (2R) a été confirmée. / Transketolase (TK, EC 2.2.1.1) catalyzes the formation of D-threo ketoses from (2R)-α-hydroxyaldehydes by the stereospecific formation of a C-C bond. Our aim was to invert the enantioselectivity of TK by protein engineering in order to obtain L-erytho ketoses (sought after for their potential pharmaceutical and/or nutritional applications) from (2S)-α-hydroxyaldehydes. For that purpose, a thermostable TK from thermophilic bacterium Geobacillus stearothermophilus (mTKgst) has been identified and overexpressed. After the study of the 3D-structure of mTKgst, two residues located in its active site (Leu382 and Asp470) were selected as mutation targets for the inversion of the enzyme’s enantioselectivity. Both rational and semi-rational approaches were considered for the construction of the mutant mTKgst libraries. In the former, the two residues were modified by site-saturation mutagenesis. In the latter, short sequences of five amino acids, neighboring target ones, were modified using a cassette mutagenesis technique. A novel continuous pH-based assay has been developed for the high-throughput screening of the mTKgst libraries, using phenol red as pH indicator. The screening revealed mTKgst-L382D/D470S as the top mutant, showing a 5-fold activity improvement towards a model (2S)-hydroxyaldehyde and the loss of enantioselectivity towards the (2R)-aldehyde.

O objetivo deste estudo foi aumentar o banco de dados em relação a resíduos (cake) e efluentes (licor) industriais e o seu nível de clastogenicidade. Este estudo contribuiu para mostrar: a) que o bioensaio com Tradescantia pallida foi sensível para a avaliação da clastogenicidade em mistura complexa de resíduos catalíticos industriais, nunca testados anteriormente. b) a tendência de uma dose resposta para ambos os resíduos catalíticos c)a pasta (cake) apresenta maior clastogenicidade que o licor nas concentrações estudadas. Provavelmente isto se deve a menor concentração de Ti e Al no licor do que no cake. / The aim of this study was to increase data concerning liquid effluent (liquor) and solid waste (cake) and their level of clastogenicity using TradMCN. This study contributed to show a) bioassay Trad-MCN with Tradescantia pallida was sensitive to evaluate the clastogenicity in a complex waste mixture, never tested before b) a tendency of a dose response for both catalytic wastes. c) higher clastogenicity of cake comparing to liquor effluent in concentrations evaluated. Probably this is due to the much lower Ti and Al concentrations in the liquor than in the cake

Indiana University-Purdue University Indianapolis (IUPUI) / DNA double strand break (DSB) is one of the most threatening of all types of DNA damages as it leads to a complete breakage of the chromosome. The cell has evolved several mechanisms to repair DSBs, one of which is break-induced replication (BIR). BIR repair of DSBs occurs through invasion of one end of the broken chromosome into a homologous template followed by processive replication of DNA from the donor molecule. BIR is a key cellular process and is implicated in the restart of collapsed replication forks and several chromosomal instabilities. Recently, our lab demonstrated that the fidelity of DNA synthesis associated with BIR in yeast Saccharomyces Cerevisiae is extremely low. The level of frameshift mutations associated with BIR is 1000-fold higher as compared to normal DNA replication. This work demonstrates that BIR stimulates base substitution mutations, which comprise 90% of all point mutations, making them 400-1400 times more frequent than during S-phase DNA replication. We show that DNA Polymerase δ proofreading corrects many of the base substitutions in BIR. Further, we demonstrate that Pif1, a 5’-3’ DNA helicase, is responsible for making BIR efficient and also highly mutagenic. Pif1p is responsible for the majority of BIR mutagenesis not only close to the DSB site, where BIR is less stable but also at chromosomal regions far away from the DSB break site, where BIR is fast, processive and stable. This work further reveals that, at positions close to the DSB, BIR mutagenesis in the absence of Pif1 depends on Rev3, the catalytic subunit of translesion DNA Polymerase ζ. We observe that mutations promoted by Pol ζ are often complex and propose that they are generated by a Pol ζ- led template switching mechanism. These complex mutations were also found to be frequently associated with gross chromosomal rearrangements. Finally we demonstrate that BIR is carried out by unusual conservative mode of DNA synthesis. Based on this study, we speculate that the unusual mode of DNA synthesis associated with BIR leads to various kinds of genomic instability including mutations and chromosomal rearrangements.

Master of Science / Genetics Interdepartmental Program / Bikram S. Gill / TILLING (Targeting Induced Local Lesions IN Genomes) is a high-throughput, reverse genetics strategy for scanning mutagenized populations for point mutations in loci of interest. Originally, TILLING was used to investigate gene function in Arabidopsis and has since been similarly applied for gene functional analysis in other organisms. TILLING also allows the generation of novel genetic variation in specific genotypes and, thus, has been implemented as a tool for crop improvement. Ethyl methanesulfonate (EMS) is a widely used mutagen to induce point mutations in most TILLING protocols. M1 plants are then self-pollinated and M2 seed harvested. A single seed is grown from each M2 progeny and tissue taken for DNA isolation. M3 seed is cataloged. DNA is pooled to increase the efficiency and aid in mutation detection. Polymerase chain reaction (PCR) is used to amplify a locus of interest using the M2 DNA pools as a template. The PCR products are digested with an endonuclease that cleaves mismatched, mutant DNA, and the digested products are visualized. The pools for which PCR products are positive for a mutation are deconvoluted to determine which individual plant of the pool was responsible for the mutation. DNA from the positive individual is sequenced to determine the type of mutation (missense, nonsense, synonymous). Individuals with mutations that are more likely to disrupt gene function (nonsense and certain missense) are studied further by growing the corresponding M3 generation. In bread wheat, Triticum aestivum, TILLING is complicated by polyploidy: genes that have homoeologs require that the functionality of each be studied. If functional homoeologs are present for all three genomes, mutants must be identified for each homoeolog, followed by successive intercrossing to produce a triple mutant plant. As a model for wheat genetics, we propose TILLING in diploid wheat. EMS mutant populations were created in diploid wheat (Triticum monococcum ssp. monococcum) and the hexaploid bread wheat cultivar ‘Jagger’. The diploid and hexaploid wheat populations were screened for mutations at the waxy locus, GBSS1, as a validation of our population and for comparative analysis of mutation rates in 2x and 6x wheat. For diploid wheat, GBSSI was screened in 716 M2 plants, and one mutant was found for 1.9 Mb screened. 3 For hexaploid wheat, GBSSI was screened in 518 M2 plants, and 30 mutants were identified within a total of 657 Kb screened, giving a mutation frequency of one mutation per 22 Kb. The reasons for this vast difference in mutation frequency between diploid and hexaploid wheat are discussed. The diploid wheat population was further examined by screening for mutations within four lignin biosynthesis candidate genes, for a total of 2 Mb screened. A single mutant was discovered for both of the lignin genes PAL6 and HCT, giving a mutation frequency of one mutation per 1 Mb screened.