The glpD gene, which encodes aerobic sn-glycerol 3-phosphate dehydrogenase, and the glpR gene, which encodes a repressor that negatively regulates the expression of the g/p regulon, map near minute 75 on the linkage map of Escherichia coli K-12. In the present study, the nucleotide sequence of the 2895 base pair of DNA containing the glpD control region and the glpE, glpG, glpR genes was determined. The translation initiation codons with adjacent ribosome-binding sites were found for these four genes. The transcription start site of the glpD gene was identified 42 base pairs upstream from the proposed methionine start codon, preceded by a region containing typical -10 and -35 sequences found in bacterial promoters. A binding site for the cyclic AMP-cAMP receptor protein complex was located just upstream from the -35 sequence, centered at position -63. The interaction site for the glp repressor was identified by using DNase I footprinting. This region contained two tandemly repeated sequences which started at the -10 sequence and continued to position +38. The glp repressor contained 252 amino acid residues and had a molecular weight of 28,046 which was deduced from the nucleotide sequence. The position of the initiation codon was verified by determination of the amino acid sequence of the N-terminus of the purified gip repressor. The presumptive helix-turn-helix region of the repressor was located near the N-terminus (amino acids 22 to 41) at a poSition analogous to that found for the operator binding domain of other repressors such as the deo and Jac repressors. The recognition helix of the glp repressor and the nucleotide sequence of the glp operator were very similar to those of the deo system. The presumptive glpR recognition helix was changed to the deoR recognition helix and the sixth amino acid arginine of the recognition helix was changed to alanine by site-directed mutagenesis. The mutant forms of the repressor had a greatly reduced affinity for the glpD operators in vivo, determined by measuring β-galactosidase activity in a strain carrying a glpD-lacZ fusion. / Ph. D.

E. coli 0157:H7 is an important foodborne pathogen, responsible for several outbreaks of hemorrhagic colitis where improperly cooked hamburger meat was thought to be the vehicle. Various time/temperature combinations were used to determine the optimum conditions of heat shock which would result in the greatest number of survivors to a 55°C heat treatment. The optimum conditions were 42°C for 5 minutes and were used throughout the study. Heat shock of aerobically grown cells resulted in an increase in the mean D value after a 55°C heat treatment by a factor of 2.1 over nonheat-shocked controls. Heat shock of anaerobically grown cells also resulted ina significant increase in mean D value over nonheat-shocked controls. Anaerobic growth itself resulted in an increase in the ability of the cells to survive the 55°C heat treatment when compared with aerobically grown cells. Both heat-shocked and anaerobically grown cells contained a protein corresponding to a sigma³² subunit of RNA polymerase which has been identified as the 71,000 Galton heat shock protein characteristic of E. coli cells. Anaerobic plating resulted in a significant increase in the mean D values of both aerobically grown and anaerobically grown cells. The largest increase in mean D values was observed in aerobically grown non-heat-shocked cells, which increased by a factor of 2.3 when plated anaerobically instead of aerobically. The activities of catalase and superoxide dismutase in aerobically grown and anaerobically grown cells were studied to determine the reason why anaerobic plating enhanced recovery of cells. The activities of both enzymes were eliminated after heat treatment at 55°C for 20 minutes, regardless of whether the cells were heat-shocked or not. The ability of heat shock and anaerobic growth to protect the cells from a subsequent heat treatment was tested by measuring the rate of release of cell materials during heating at 55°C. Heat-shocking and anaerobic growth resulted in even faster release of cell materials during heating than controls, suggesting that neither of these stresses protected the cells against the effects of heat. The effect of heat shock on cell injury was studied. Heat shock of aerobically grown cells resulted in the greatest difference in log number of cells between cells plated in nonselective medium vs. selective medium. Thus, more cells were injured if heat-shocked than if not heat-shocked. Heat-shocking of anaerobically grown cells also resulted in more injured cells than non-heat-shocked controls. / Ph. D.

Ribonuclease II (RNase II) is a bacterial enzyme responsible for 90% of the exonucleolytic degradation of mRNA in bacteria, and has bacterial homologues known to be involved in virulence. The goal of this project was to examine the structural dynamics of RNase II using fluorescence. Prior to the beginning of this project, little was known regarding the structural composition of RNase II – required information in the study of structural dynamics. Consequently, the structure of RNase II was studied by constructing a series of deletion mutants in order to map the domains. The publication of an atomic resolution structure of RNase II allowed the project to move directly into the study of RNase II structural dynamics as it degrades mRNA. As a step towards this, RNase II was fluorescently labeled, and preliminary binding studies of DNA – a competitive inhibitor – to RNase II using fluorescence were conducted. / xii, 90 leaves : ill. (some col.) ; 29 cm

The proteins required for catabolism of glycerol 3- phosphate are encoded by the genes of the <i>glp</i> regulon of Escherichia coli and are under negative transcriptional regulation by the <i>glpR</i>-encoded repressor. The <i>glpR</i> gene is adjacent to, and is transcribed divergently from, <i>glpD</i>. <i>GlpR</i> and <i>glpD</i> are separated by two open reading frames, designated <i>glpE</i> and <i>glpG</i>, encoding proteins of unknown function. The <i>glpD</i>-encoded aerobic sn-glycerol 3-phosphate dehydrogenase is a cytosplasmic membrane-associated respiratory enzyme. The nucleotide sequence of <i>glpD</i> was determined. An open reading frame of 501 codons was preceded by a consensus Shine-Dalgarno sequence. The proposed translational start and reading frame of glpD were confirmed by determining the nucleotide sequence across the fusion joint of a <i>glpD-lacZ</i>- translational fusion. / Ph. D.

The first reports of bacteria as a cancer therapy date back to the pioneering work of Dr. William Coley–now widely regarded as the father of immunotherapy. As far back as 1891, Coley demonstrated that the intratumoral injection of live and later heat-killed isolates of Streptococcus pyogenes and Serratia marcescens could induce durable remission in patients with bone and soft tissue sarcoma. While this therapy was deemed to unsafe at the time, Coley’s findings have formed the basis for our modern understanding of immunology and cancer immunotherapy. Over the past two decades, the advent of synthetic biology is driving a new era of medicine through the genetic programming of living cells. This transformative approach enables the creation of engineered systems that sense and respond to diverse environments, permitting safe and effective targeted delivery of therapeutic payloads within disease sites. In this thesis, I seek to utilize principles from synthetic biology and immunology to engineer bacteria for immunotherapeutic delivery. I have developed multiple strains of non-pathogenic E. coli capable of colonizing solid tumors and delivering diverse immunotherapeutic payloads specifically within the tumor. This local delivery approach enables the utilization of therapeutic agents that may be otherwise systemically toxic. In one instance, we engineered an encoded nanobody antagonist of CD47 (CD47nb), an anti-phagocytic receptor commonly overexpressed in several human cancers. We show that delivery of CD47nb by tumor-colonizing bacteria increases activation of tumor-infiltrating T cells, stimulates rapid tumor regression, prevents metastasis, and leads to long-term survival in a syngeneic tumor model. Thus, engineered bacteria may be used for safe and local delivery of diverseimmunotherapeutic payloads leading to systemic antitumor immunity.

Photoactivated psoralens and other agents that form DNA interstrand crosslinks are highly cytotoxic and are useful in treating a range of diseases, including vitiligo, psoriasis, and some forms of cancer. Unlike many lesions that damage only one strand of the duplex DNA, DNA interstrand crosslinks form covalent bonds with both strands. Thus, repairing these lesions is complicated both by the lack of an undamaged strand to serve as a template for resynthesis following excision, as well as the potential to form double strand breaks if both strands are incised. A number of models have proposed that repair is likely to couple nucleotide excision repair with other repair pathways such as recombination, and/or translesion synthesis. However, several aspects of these models remain speculative, and how these medically relevant lesions are repaired by cells still remains elusive. In this study, I use Escherichia coli as a model organism to characterize which gene products contribute to survival in the presence of psoralen-induced DNA interstrand crosslinks. In Chapter II, I demonstrate that although nucleotide excision repair initiates repair, not all subunits contribute equally to survival. Notably, uvrC is less sensitive to psoralen-induced damage than either uvrA or uvrB. I found that Cho, an alternative endonuclease, accounts for the increased resistance of uvrC mutants and contributes to survival in the presence of UvrABC. Cho was not required following angelicin treatment, a psoralen derivative that only forms monoadducts, suggesting that Cho function is specific for interstrand crosslink repair. However, Cho, by itself, is not required for the initial incision and only modestly enhances the rate that psoralen crosslinks are incised in vivo. Following incision, many of the intermediates in the repair process remain speculative. In Chapter III, I examine how recombination and translesion synthesis mutants contribute to survival of psoralen-induced damage. I show that both recBC and recF contribute to survival, but that neither mutant is as hypersensitive as recA, potentially suggesting that pathways involving either single strand gaps or double strand break intermediates can occur during repair. Finally, I show that Polymerase V is responsible for the translesion synthesis that contributes to survival in the case of psoralen-induced damage in E.coli.

Synthetic biology enables the engineering of interactions between living medicines to overcome the specific limitations of monotherapies. A major challenge facing tumor-antigen targeting therapies like chimeric antigen receptor (CAR)-T cells is the identification of suitable targets that are specifically and uniformly expressed on heterogeneous solid tumors. In contrast, certain strains of bacteria are gaining recognition as a new class of antigen-agnostic cell therapy due to their selective growth within the immunosuppressive niche of the solid tumor microenvironment (TME). In response, this dissertation aims to pair the cytotoxicity of CAR-T cells with the antigen-independent specificity of tumor-colonizing bacteria to create a new strategy for solid tumor recognition. Here, we reprogram the probiotic strain of E. coli Nissle 1917 to release synthetic CAR targets and human chemokines directly within the solid tumor core. To enable universal targeting, we design synthetic targets to bind ubiquitous components of the TME and broadly tag tumor tissue for CAR-mediated lysis. We demonstrate that these targets robustly coat the surface of cancer cell lines and lead to effective killing by CAR-T cells across various cancer types. We additionally show that injected probiotics selectively grow within the tumor core and maintain target production ¬ in situ – leading to therapeutic efficacy across multiple genetically distinct tumor models. Within this dissertation, we also reveal that intratumoral bacteria provide natural adjuvant effects that serve to activate and increase the effector functions of CAR-T cells in vivo. However, we discover that this can lead to early T cell exhaustion and terminal effector differentiation. To mitigate the counterproductive effects of overstimulation, we generate a new probiotic strain with reduced inflammatory properties that significantly improves CAR-T cell phenotype – leading to enhanced therapeutic benefit in a human model of leukemia. We conclude by discussing the numerous avenues available to optimize cross-Kingdom signaling and to ultimately leverage the full therapeutic benefit of combined cell therapies for future translation. Altogether, this dissertation highlights the potential of the probiotic-guided CAR-T cell (ProCAR) platform to address the critical roadblock of identifying suitable CAR targets by providing an antigen in situ that is orthogonal to both healthy tissue and tumor genetics – and, in turn, aims to establish the foundation for engineered communities of living medicines.

Indiana University-Purdue University Indianapolis (IUPUI) / The multi-protein complex of F0F1 ATP synthase has been of great interest in the fields of microbiology and biochemistry, due to the ubiquitous use of ATP as a biological energy source. Efforts to better understand this complex have been made through structural determination of segments based on NMR and crystallographic data. Some experiments have provided useful data, while others have brought up more questions, especially when structures and functions are compared between bacteria and species with chloroplasts or mitochondria. The epsilon subunit is thought to play a signi cant role in the regulation of ATP synthesis and hydrolysis, yet the exact pathway is unknown due to the experimental difficulty in obtaining data along the transition pathway. Given starting and end point protein crystal structures, the transition pathway of the epsilon subunit was examined through computer simulation.The purpose of this investigation is to determine the likelihood of one such proposed mechanism for the involvement of the epsilon subunit in ATP regulation in bacterial species such as E. coli.