Celiac disease is an autoimmune enteropathy resulting from the ingestion of gluten and gluten-like proteins from wheat, barley and rye in afflicted individuals. Indigestible gluten-derived peptides rich in proline residues are known to be responsible for eliciting the inappropriate immune response characteristic of the disease. In this investigation, surface level expression of prolyl peptidase activity by genetically engineered probiotic lactobacilli was postulated to be a possible treatment for this disease. Plasmid-based reporter vectors were constructed utilizing a novel, homology-based cloning technique to assess the expression and localization signals from the S-layer protein gene of Lactobacillus acidophilus. These plasmids were mutated during construction due to toxicity associated with the cloned cassettes. The toxicity of the Slayer secretion and/or anchoring domains in E. coli was confirmed by cloning the fused components into an inducible expression system. When the prolyl peptidase, Xaa-Pro, from L. reuteri was incorporated into the S-layer expression cassette, the full-length protein was efficiently expressed but was not active, likely due to protein aggregation and inclusion body formation. Future research directions are discussed and a modified experimental design strategy is presented. This work provides a foundation for continued investigation into the feasibility of utilizing genetically engineered lactobacilli as a potential treatment strategy for celiac disease.

Antimicrobial resistance development is a global concern. A handful of effective treatments are currently available for controlling bacterial disease in animal and plant systems. In humans, overuse of antibiotics poses a serious threat. In the United States, over two million nosocomial infections are reported annually for which traditional antibiotic treatments are often ineffective, requiring a high-risk aggressive treatment of cocktail antibiotics. Copper (Cu)-based biocides are currently the standard for controlling bacterial and fungal diseases of many crops. However, there is an increasing concern of resistance development and non-target environmental toxicity of Cu biocides to aquatic species and other beneficial organisms. To reduce Cu input in the environment and combat against Cu resistance, high-performing Cu or Cu alternatives must be developed. In this dissertation research, a few novel alternatives to traditional antimicrobial compounds (antibiotics and Cu) were developed and studied. A composite of Gallium and N-Acetyl Cysteine (Ga-NAC) as an alternative to conventional antibiotics was studied for treating Pseudomonas aeruginosa infections. The Ga-NAC composite treatment revealed strong anti-biofilm properties, exhibiting a high therapeutic window. Improved Ga bioavailability at the cell surface is linked to enhanced intracellular uptake and killing. Two novel composite materials were developed against Xanthomonas citri subsp. citri, a pathogen responsible for citrus canker disease. A composite of zinc oxide and nano-copper (ZnO-n-Cu) was developed to reduce Cu application rate. Performance of the ZnO-n-Cu composite in field conditions was evaluated in a commercial grove for two consecutive years. The ZnO-n-Cu composite suppressed citrus canker disease at five times lower copper concentration in comparison to Cu standards. Another composite material of silica gel and quaternary ammonium compound (Fixed-Quat) was developed and tested as a non-metal alternative to copper biocide. Fixed-Quat demonstrated similar field efficacy against citrus canker when compared to Cu standards for three consecutive seasons.

<p>Secondary metabolites produced by bacterial species serve many clinically useful purposes such as anti-bacterial, anti-cancer, and immunosuppresive agents. Actinobacteria, particularly the genus <em>Streptomyces</em>, have been an abundant source of such metabolites for the past half century. The production of secondary metabolites is controlled through vast regulatory cascades, but the activation and control of these pathways is still poorly understood. This leads to the inability to isolate all of the secondary metabolites that <em>Streptomyces</em> are capable of producing. This study focuses on the comparison of synthetic small molecules, which were found to alter the production of secondary metabolites in <em>S. coelicolor</em>. A comparative analysis of two of these molecules, ARC2 and ARC6, shows they modulate secondary metabolites in different ways. In a separate study, ARC2 was shown to achieve this phenotype through the inhibition of a target in fatty acid biosynthesis. The results of this study suggest that ARC6 does not have the same target, although it may target the same metabolic system. Furthermore, these two molecules also have opposite effects on <em>S. coelicolor </em>development. The cumulative results of this study suggest that ARC2 and ARC6 can act as separate chemical tools in enhancing the understanding of secondary metabolism.</p> / Master of Science (MSc)

No description available.

Chromosomally determined arsenate resistance in Escherichia coli is well characterized. Little, however, is known about chromosomally determined arsenite resistance in E. coli. Accordingly, spontaneous arsenite resistant mutants were selected in a plasmid free strain of E. coli. One mutant strain was analyzed in detail, genetically and physiologically. The mutation confering arsenite resistance was shown to be a single gene mutation. Genetic mapping studies using conjugation and transduction showed that the mutation was closely linked to the ilv region of the E. coli map.

Soil bacteria and fungi play a central role in the biogeochemical cycling of both carbon (C) and nitrogen (N) through terrestrial ecosystems. In the C cycle, soil microbial groups regulate the depolymerization of large stocks of soil organic matter and contribute 35-69 Pg C to the atmosphere annually through heterotrophic respiration. Soil microbial groups also mediate several important transformations of N, including making limiting nutrients available for uptake by plants through N-fixation, converting N between inorganic forms through nitrification, and returning N to the atmosphere through denitrification. While each of these functions is performed by soil microbes, scaling microbial physiology and community structure to biogeochemical cycling remains a significant research challenge. This dissertation integrates three distinct approaches to characterizing relationships between microbial physiology, microbial community structure and biogeochemical cycling. First, I explore the role of microbial physiology in C cycling by developing a novel method to predict bacterial carbon use efficiency (CUE) from genomes using metabolic modeling. I find that bacterial CUE is phylogenetically structured, with the class and order levels explaining the greatest proportion of variance in CUE, and I identify particular bacterial traits that most strongly predict CUE. These findings highlight the importance of accounting for microbial physiology when modeling soil C cycling. Second, I explore how differences in the abundance and activity of microbial functional groups and their interactions with mycorrhizal fungi impact temperate forest N cycling. I find that N availability and rates of N-fixation, nitrification and denitrification are structured in relation to mycorrhizal fungal types, but that the abundances of bacterial functional groups are not correlated with biogeochemical fluxes. Finally, I use a soil biogeochemical model to identify sources of uncertainty and data needs in advancing our understanding of microbially-mediated soil biogeochemical cycling. I isolate specific microbial physiological and enzyme kinetic parameters that have disproportionately large impacts on projections of coupled C and N cycling, and I quantify the potential for particular types of data to help reduce uncertainties. Overall, this dissertation advances our understanding of how microbial processes impact the biogeochemical cycling of C and N in terrestrial ecosystems.

Rhizobium leguminosarum is a gram-negative soil bacterium that requires iron for survival. However, iron becomes insoluble in the presence of oxygen at physiological pH. In response, Rhizobia species have used siderophore mediated iron transport systems to meet their iron requirements. R. leguminosarum ATCC 14479 produces the trihydroxymate siderophore vicibactin and we hypothesize that the import of the ferric iron-vicibactin complex is energized by the TonB-ExbB-ExbD system. Here, we have identified a putative tonB gene. A tonB mutant was created and compared with wild type in its ability to transport 55Fe-vicibactin. Also, the putative TonB of R. leguminosarum ATCC 14479 is interesting due to its estimated size compared to the TonB of E. coli.. Many groups have attempted structural analysis of the C-terminus of TonB in E. col with inconsistent results. We were successful in expressing 2 different sized TonB C-terminals (120 and 200 amino acids) using pET17b in E. coli.

Many endospore-forming bacteria cause diseases such as anthrax and food poisoning. Spores however also contribute to various agricultural and industrial processes. Spores possess extreme resistance properties, notably to chemical, et and dry heat, desiccation, and UV damage. For pathogenic spore formers, this poses an issue as spores are resistant to most decontamination methods currently in use. This work focuses on characterizing proteins thought to contribute to spore stability and efficient spore germination. Understanding how spores can remain stable for long periods of dormancy and against various insults and rapidly initiate germination could allow for the development of techniques that induce germination early and rapidly, promoting inexpensive decontamination. Physiological studies found that a family of spore-associated lipoproteins is needed for efficient spore germination and influences membrane fluidity in dormant spores. All the members of the lipoprotein family serve the same function, as each can fulfill the role of another. In vivo cross-linking was used to characterize protein-protein interactions found on the inner spore membrane. Glutaraldehyde crosslinking revealed that the four lipoproteins appear to interact. Bacterial two-hybrid analysis on individual protein domains further suggests the lipoproteins seem to interact through their predicted ring-building motif within their otherwise uncharacterized domains. Additionally, the absence of the spore lytic enzyme SleB seems to alter the crosslinking pattern of the lipoproteins, suggesting either it's interacting or helping facilitate lipoprotein interactions. Fluorescence microscopy reveals an unequal spatial distribution of the lipoproteins on the spore membrane, which seems to be supported by preliminary super-resolution microscopy studies. Further work aiming to characterize the entire inner spore membrane interactome is currently being conducted. The presented research used many methods and built many collaborations with the goal of providing insight to spore dormancy and efficient spore germination with an additional goal of understanding inner spore membrane protein behavior and how it leads to the highly resistant properties native to bacterial endospores. / Doctor of Philosophy / Certain species of Gram-positive bacteria can form a dormant cell called a bacterial endospore. Endospores, or spores, are highly resistant to insults such as noxious chemicals, wet and dry heat, and UV irradiation. These resistance properties make spores immune to standard sanitation methods, and result from various anatomical structures innate to the spore. In medicine, this poses a problem as spore forming bacteria can be causative agents of diseases such as food poisoning, anthrax, infant botulism, hospital-acquired diarrhea, and others. In agriculture and other industries, spore forming bacteria can be used as insecticides or fungicides, and have a long shelf life, making them ideal for long term storage. Research in the following document aims to answer questions relating to how spores transition from dormant spore to a typical cell. Understanding of these processes can inform novel decontamination techniques, better more stable spore-based products, and subversion of disease, depending on which process/ structure in the spore is altered.

Due to high rates of emerging antibiotic resistant bacteria, research into combatting these “superbugs” has focused on engineering more effective treatments for bacterial infections, including pairing antibiotics having varied mechanisms of action to elicit a stronger antimicrobial response than a single antibiotic treatment. Previous studies have characterized pairwise antibiotic interactions as having synergistic, additive, or antagonistic relationships, suggesting the possibility that many factors influence the resulting response. As an extension of the research done on paired antibiotic interactions, this work endeavored to examine whether changing nutritional environments had an effect on documented synergistic relationships between various groups of antibiotics. To corroborate previous studies, antibiotics from the β lactam (targeting cell wall synthesis) and aminoglycoside (targeting protein synthesis) families were chosen as test subjects and paired with antibiotics from other classes under varied nutrient conditions. Streptomycin (STR) and Amikacin (AMK) were two aminoglycosides tested in their relationship to Ciprofloxacin (CPR), a fluoroquinolone (targeting DNA replication). The relationships between the aminoglycosides and CPR remained consistently synergistic in various media, including two complex, undefined media, LB and yeast extract (YE), and dilution of both media with a saline solution to half-strength. In contrast, the selected β lactams Piperacillin (PIP) and Ampicillin (AMP) did not show a previously documented synergy with the macrolide (targeting protein synthesis) Erythromycin (ERY), and alterations to the culture medium vastly affected the PIP/ERY combined relationship. Collectively, these results suggest that antibiotic interactions are not based on a singular mechanism, and that these relationships may be able to be manipulated through environmental factors such as changing nutritionalenvironments. A multi-faceted pathway for manipulating drug interactions may be key in increasing the efficacy of antibiotic treatment plans.

Porphyromonas gingivalis, a gram-negative anaerobic, pathogenic bacterium is a major etiological agent in the initiation and progression of periodontal disease. Due to the ever-changing environment of the oral cavity, inhabitants like Porphyromonas gingivalis must possess the ability to adapt to changes in environmental conditions like pH, temperature, oxygen tension, and metal concentration. P. gingivalis should therefore have an efficient regulatory system in order to adapt and survive in the oral cavity. This response adaptation occurs at the transcriptional level, which involves alternative sigma factors. Extracytoplasmic function sigma (ECF-s) factors are the largest group of alternative sigma factors that play a role in the bacterial response to environmental stress conditions. Here we analyze the s-70 factor gene, PG0214, an extracytoplasmic function sigma factor encoded in the P. gingivalis genome, and examine its role in the bacterial response to environmental stress and virulence. Our findings indicate that the PG0214 gene is important in regulating major functional gene groups and pathways in the P. gingivalis genome. Strains deficient in the PG0214 gene were analyzed and shown to have decreased protease activity, as well as reduced survivability and invasion rates in eukaryotic host cells when compared against wild-type W83 and ATCC 33277 strains. Collectively our studies demonstrate that the PG0214 gene is a positive regulator of gene expression for the survival and virulence of P. gingivalis in the presence of oxidative- and iron-stress, although further study is needed to fully characterize the gene and determine its specific function.