Allosteric regulation of proteins by reversible ligand binding is essential for regulation of fundamental biological processes. The mechanism by which a binding event alters the function of a distant site in a protein is only poorly understood. In this thesis, I use the Tetracycline Repressor (TetR) as a model system to study ligand induced allostery. The transcription of genes encoding the resistance to the antibiotic, tetracycline (Tc), is repressed by TetR, which is a homodimeric alpha-helical protein possessing a small N-terminal DNA binding domain (DNB domain) and a larger C-terminal tetracycline binding and dimerization domain (TBD domain). Based on previous structural and thermodynamic studies, the DNB domains are thought to exist in two stable, distinct conformations. One conformation is able to bind the Tc resistance operator sequence (tetO) with high affinity, while the other, which is induced by Tc binding, binds very weakly. While most previous studies on TetR have focused on the effects of Tc binding on the DNB domain conformation, here I have investigated the role of the DNB domain in modulating Tc binding. By introducing destabilizing mutations into the DNB domain I ascertained that the conformation and stability of the DNB domain plays an important role in determining Tc binding affinity. I also discovered that in the absence of ligand, the DNB domain exists in an unstable and flexible state with respect to the TBD domain. However, Tc binding to the TBD domain stabilizes the DNB domain, causing it to fold cooperatively with the TBD domain. I have discovered that the behavior of previously isolated non-inducible mutants is caused by the inability of Tc to stabilize the DNB domain in these mutants. Furthermore, reverse TetR mutants, which bind DNA better in the presence of Tc have an unfolded DNB domain that is only partially stabilized by Tc binding. My work suggests a new comprehensive, Tc induced stabilization and domain cooperativity model that can describe the mechanism of allostery in TetR and previously unexplainable mutants. A practical outcome of this research is the creation of a Tc induced folding switch that can be exploited to control the in vivo degradation of a protein of interest.

Allosteric regulation of proteins by reversible ligand binding is essential for regulation of fundamental biological processes. The mechanism by which a binding event alters the function of a distant site in a protein is only poorly understood. In this thesis, I use the Tetracycline Repressor (TetR) as a model system to study ligand induced allostery. The transcription of genes encoding the resistance to the antibiotic, tetracycline (Tc), is repressed by TetR, which is a homodimeric alpha-helical protein possessing a small N-terminal DNA binding domain (DNB domain) and a larger C-terminal tetracycline binding and dimerization domain (TBD domain). Based on previous structural and thermodynamic studies, the DNB domains are thought to exist in two stable, distinct conformations. One conformation is able to bind the Tc resistance operator sequence (tetO) with high affinity, while the other, which is induced by Tc binding, binds very weakly. While most previous studies on TetR have focused on the effects of Tc binding on the DNB domain conformation, here I have investigated the role of the DNB domain in modulating Tc binding. By introducing destabilizing mutations into the DNB domain I ascertained that the conformation and stability of the DNB domain plays an important role in determining Tc binding affinity. I also discovered that in the absence of ligand, the DNB domain exists in an unstable and flexible state with respect to the TBD domain. However, Tc binding to the TBD domain stabilizes the DNB domain, causing it to fold cooperatively with the TBD domain. I have discovered that the behavior of previously isolated non-inducible mutants is caused by the inability of Tc to stabilize the DNB domain in these mutants. Furthermore, reverse TetR mutants, which bind DNA better in the presence of Tc have an unfolded DNB domain that is only partially stabilized by Tc binding. My work suggests a new comprehensive, Tc induced stabilization and domain cooperativity model that can describe the mechanism of allostery in TetR and previously unexplainable mutants. A practical outcome of this research is the creation of a Tc induced folding switch that can be exploited to control the in vivo degradation of a protein of interest.

The continuing emergence of multi-drug resistant Mycobacterium tuberculosis is threatening the ability to treat tuberculosis (TB) worldwide. The development of new anti-TB drugs requires new approaches and new drug targets. In this study, a mariner-based transposon, TnQuoVadis, was constructed to identify essential genes as potential drug targets. This transposon has an outward-facing anhydrotetracycline (ATc)-inducible promoter at each end. A mutant with TnQuoVadis inserted upstream of an essential gene may display normal growth in the presence of ATc, but exhibit no growth or severely diminished growth in the absence of ATc. TnQuoVadis was placed onto a vector with a temperature sensitive replication origin for more efficient mutagenesis of mycobacteria. In a preliminary genetic screen using the model organism Mycobacterium smegmatis, 13 mutants with ATc-dependent growth were identified. Identification of the insertion sites by cloning and sequencing indicated that there were nine genetic loci containing transposon insertions upstream of essential gene candidates in M. smegmatis. Further analysis of these genes indicated that many were previously known essential in both M. smegmatis and M. tuberculosis. These results demonstrate that TnQuoVadis and its delivery system can be utilized for the identification of essential genes in mycobacteria / Master of Science

Synthetic biology views a complex biological system as an ensemble in the hierarchy of parts, devices, systems, and networks. The practice of using engineering rules such as decoupling and standardization to understand, predict, and re-build novel biological functions from model-driven designed genetic circuits is emphasized. It is one of the top ten technologies that could help solving the current and potential risks in human society. Cyanobacteria have been considered as a promising biological system in conducting oxygenic photosynthesis to convert solar energy into reducing power, which drives biochemical reactions to assimilate and generate chemicals for a specific purpose such as CO2 fixation, N2 fixation, bioremediation, or fuels production. The promoter is a key biological part to construct feedback loops in genetic circuits for a desired biological function. In this thesis, promoters that don't work in the cyanobacterium Synechocystis PCC 6803 in terms of promoter strength, and dynamic range of gene regulation are identified. Biological parts, such as ribosome binding sites, and reporter genes with and without protease tags were also characterized with the home-built broad-host-range BioBrick shuttle vector pPMQAK1. The strong L03 promoter, which can be tightly regulated in a wide dynamic range by the foreign Tet repressor, was created through an iterative promoter engineering cycle. The iteration cycle of DNA breathing dynamic simulations and quantification of a reporting signal at a single-cell level should guide through the engineering process of making promoters with intended regulatory properties. This thesis is an essential step in creating functional promoters and it could be applied to create more diverse promoters to realize the emphasized practices of synthetic biology to build synthetic cyanobacteria for direct fuel production and CO2 assimilation.

Antibiotic resistance is a worsening threat to human health. Increasing our understanding of the mechanisms causing this resistance will be of great benefit in designing methods to evade resistance and in developing new classes of antibiotics. In this thesis, I have used the TetR Family Transcriptional Regulators (TFRs), which constitute one of the largest antibiotic resistance regulator families, as a model system to study the structure, function and evolution of antibiotic resistance determinants. I performed a thorough examination of the variation and conservation seen in TFR sequences and structures using computational approaches. Through structure comparison, I have identified the most conserved features shared by the TFR family that are crucial for their stability and function. Based on my findings on conserved TFR structural features, a quantitative assay of binding affinity determination was developed. Through sequence comparison and a residue contact map method, I discovered the existence of a conserved residue network that correlates well with the known allostery pathway of TetR. This predicted allosteric communication network was experimentally tested in TtgR. I have also developed methods to identify TFR operator sequences through genomic comparisons and validated my prediction through experiments. In addition, I have developed an in vivo system that can be used to identify and characterize proteins that mediate resistance to almost any antibiotic. This system is simple, fast, and scalable for high-throughput applications, and could be used to discover a wide range of novel antibiotic resistance mechanisms. The principles that I applied to the TFR family could also be applied to other protein families.

The ability to control expression of a gene of interest is an important tool of molecular biologists and genetic engineers. This allows the phenotype associated with the regulated gene or genetic pathway to be partially de-coupled from the genotype and expressed only under condition that lend to induction of the genetic control system employed. Such control is typically implemented through a repressor protein (Eg. TetR, LacI) which will repress transcription when bound to a promoter containing a binding site (operator) recognized specifically by that repressor. Many such repressors and their cognate promoters are well-defined and characterized in model genetic systems, such as Escherichia coli, and may function poorly in other bacterial species. A lack of genetic components that allow the controlled expression of heterologous genes in less well studied bacterial species may limit their bio-industrial potential and the sophistication of engineered phenotypes. The work presented here uses random mutagenesis and selection to isolate mutants of TetR that are inducible by increased culture temperature. Induction of protein expression by temperature change can have benefits over repressors that require small-molecule inducers in bio-industrial applications as reversal of induction and reuse of growth medium are possible. The host range of these, or any, repressor protein is limited by the host range in which its cognate promoter will function. To bypass this limitation and allow use of TetR in Francisella novicida, a method was developed by which TetR-responsive promoters that function in this host could be selected from random DNA sequence flanking the TetR binding site, tetO. Many unique TetR-repressible promoters that function in Francisella were recovered and tightly-regulated expression of both exogenous reporter genes and host virulence genes were demonstrated. This promoter selection technique was also applied to E. coli, which allowed comparison between Francisella-selected promoters and those selected in an E. coli host. Adaption of this process for production of promoters responsive to transcription factors other than TetR would simply require the use of a different operator sequence, suggesting diverse applications for this technique. This success in promoter engineering should enable advances in synthetic biology and genetic engineering in non-model bacterial species. / Graduate

Antibiotic resistance is a worsening threat to human health. Increasing our understanding of the mechanisms causing this resistance will be of great benefit in designing methods to evade resistance and in developing new classes of antibiotics. In this thesis, I have used the TetR Family Transcriptional Regulators (TFRs), which constitute one of the largest antibiotic resistance regulator families, as a model system to study the structure, function and evolution of antibiotic resistance determinants. I performed a thorough examination of the variation and conservation seen in TFR sequences and structures using computational approaches. Through structure comparison, I have identified the most conserved features shared by the TFR family that are crucial for their stability and function. Based on my findings on conserved TFR structural features, a quantitative assay of binding affinity determination was developed. Through sequence comparison and a residue contact map method, I discovered the existence of a conserved residue network that correlates well with the known allostery pathway of TetR. This predicted allosteric communication network was experimentally tested in TtgR. I have also developed methods to identify TFR operator sequences through genomic comparisons and validated my prediction through experiments. In addition, I have developed an in vivo system that can be used to identify and characterize proteins that mediate resistance to almost any antibiotic. This system is simple, fast, and scalable for high-throughput applications, and could be used to discover a wide range of novel antibiotic resistance mechanisms. The principles that I applied to the TFR family could also be applied to other protein families.

<p>Bacteria adapt to changes in their environment by regulating gene transcription. TetR family transcriptional regulators (TFRs) constitute one of the largest groups of bacterial transcription factors and thus, characterization of TFRs is anticipated to be crucial for a better understanding of prokaryotic physiology. Of significant importance, the majority of TFRs are predicted to respond to small-molecule signals and an emerging paradigm suggests that identifying ligands of TFRs can provide direct insight into the biochemical functions of the genes they regulate. Regulatory target genes and small-molecule ligands are unknown for all but a few TFRs and therefore, generally applicable tools for identifying these basic elements of TFRs are highly desirable. We first investigated the use of genome context as a predictive tool for identifying regulatory targets of TFRs. We find that the majority of TFRs are divergently oriented from a neighboring gene, and those with a“200 bp rule” should allow us to predict at least one regulatory target for more than half of all TFRs in the public databases. Second, we developed a biosensor mechanism amenable to high-throughput screening for identifying ligands of TFRs of unknown function. Significantly, one of our biosensors has played an integral role in characterizing the ligands of a previously uncharacterized TFR. Thus, the combined use of the tools we have developed will provide considerable benefit in understanding bacterial small-molecules responses mediated by TFRs.</p> / Doctor of Philosophy (PhD)