Porphyromonas Gingivalis (P.G.) is a gram-negative anaerobic bacillus that is commonly implicated in periodontal disease in humans via invasion of oral epithelial cells. Characterizing the intracellular mechanisms that allow for these infections to take place is important for future attempts to stop or halt the spread of infection. Given the complexity of bacterial virulence, research on the subject often necessitates precise measurements of very specific biochemical pathways. In this study, we focus on the type IX secretion system utilized by P.G. to initiate colonization of host cells. Specific to this secretion system is the PorX-PorY two-component regulatory system. Here we use the bacterial adenylate cyclase based 2 hybrid system to test if two specific domains of the PorX-PorY system communicate intracellularly with each other; and hence gain further knowledge on how this infection occurs.

Methanogens anaerobically metabolize simple carbon compounds coupled with an electron donor and produce methane in a process known as methanogenesis. While their importance in anoxic ecosystems and their greenhouse gas emissions are known, less is known about their diverse members. This is in part due to limited culture-dependent studies as a consequence of the difficulty to culture and isolate them under laboratory conditions. Current methods in methanogen isolation require lengthy protocols, expensive equipment, can be easily contaminated, and even if a successful isolation is completed, traditional methods are biased towards only a few species of methanogens- leaving much of this community unsampled and thus unrepresented. New approaches in the isolation of methanogens need to be investigated in order to circumvent these obstacles. Here, I evaluated the effects of different strategies and alternative methods with the goal of increasing the diversity of recovered methanogens from Amazon peatlands as a study case. The results show that: a) through the use of different antibiotics the bacterial community makeup can be altered and lead to different methanogenic enrichments, some antibiotics reliably increase methanogenesis in all study sites, others only enhance it in some sites, while some have a low rate of methanogenesis enriching novel slow growers, b) the use of different substrates has less of an effect on methane production rates, however the complex substrate butyrate leads to consistent late stimulation, c) altering media components (reducing agent and overall geochemical background) for Amazon conditions would lead to a shorter time to isolation, d) and multiple methanogenic enrichments were achieved building on variable conditions and can lead to novel Amazon lineages. Molecular data is offering a more detailed view of bacteria and methanogens increasing or decreasing in response to treatments. Overall, it is shown that combining alternative approaches that manipulate interactions, metabolic substrate availability and culturing conditions could lead to more diverse isolation outputs from methanogenic cultures.

Porphyromonas gingivalis (P. gingivalis) is an oral pathogen known for causing periodontal diseases like periodontitis and alveolar bone loss. In this study, we investigate the molecular mechanisms of P. gingivalis with focus of the molecular cloning of the two DNA strains of the bacteria PGN_1740 and PGN_0012 in the Ampr pTCow. PGN_1740 is an RNA polymerase ECF-type sigma factor used for transcription. PGN_0012 is a two-component system regulator gene that is important in signal transduction. We demonstrated the cloning mechanism through transformation and confirmed the results through gel electrophoresis and using a positive transformant as a control. The process of cloning the DNA inserts into the bacteria followed a polymerase chain reaction for the amplification of the DNA fragments, digestion of the plasmid and DNA fragments with the restriction endonucleases (BamHI and HindIII), ligation and finally heat shock transformation are presented in this thesis. The effectiveness of these procedures was observed through agarose gel electrophoresis and ethanol precipitation for the purification of the PCR products. In this investigation, we discuss molecular and biological characterization of the P. gingivalis bacteria in regard to cloning and ampicillin resistance.

Colorectal cancer is the third most common type of cancer that affects both men and women and the second leading cause of death in cancer related deaths[1, 2]. The most common form of treatment is chemotherapy followed by radiation, which is insufficient to cure stage four cancers[3]. Salmonella enteric has long been shown to have inherent tumor targeting properties and have been able to penetrate and exist in all aspects of the tumor environment, something that chemotherapy is unable to achieve. This lab has developed a genetically modified Salmonella typhimurium (GMS) which is able to deliver DNA vaccines or synthesized proteins directly to tumor sites. These GMS strains have been used to deliver human TNF-related apoptosis inducing ligand (TRAIL) protein directly to tumor sites, but expression level was limited. It is the hope of the experiment that codon optimization of TRAIL to S. typhimurium preferred codons will lead to increased TRAIL expression in the GMS. For preliminary studies, BALB/c mice were subcutaneously challenged with CT-26 murine colorectal cancer cells and treated with an intra-tumor injection with either PBS, strain GMS + PCMV FasL (P2), or strain GMS + Pmus FasL). APC/CDX2 mutant mice were also induced to develop human colon polyps and treated with either PBS, strain GMS + vector (P1), P2, or P3. The BALB/c mouse showed statistically significant levels of decreased tumor size in groups treated with P2 or P3. The APC/CDX2 mouse study showed statistically significant levels of decreased colon polyp numbers in groups treated with P3, as expected, but was not significantly significant for groups treated with P1 and P2. In addition, TRAIL was codon optimized for robust synthesis in Salmonella. The construct will be characterized and evaluated in vitro and in vivo. Hopefully, the therapeutic effect of codon optimized TRAIL will be maximal while almost completely minimizing any unintended side effects.

The Multiple Antibiotic Resistance Regulator Family (MarR) are transcriptional regulators, many of which forms a dimer. Transcriptional regulation provides bacteria a stabilized responding system to ensure the bacteria is able to efficiently adapt to different environmental conditions. The main function of the MarR family is to create multiple antibiotic resistance from a mutated protein; this process occurs when the MarR regulates an operon. We hypothesized that different transcriptional regulator genes have interactions with each other. It is known that Salmonella pagC transcription is activated by three regulators, i.e., SlyA, MprA, and PhoP. Bacterial Adenylate Cyclase-based Two-Hybrid (BACTH) system was used to research the protein-protein interactions in SlyA, MprA, and PhoP as heterodimers and homodimers in vivo. Two fragments, T25 and T18, that lack endogenous adenylate cyclase activity, were used for construction of chimeric proteins and reconstruction of adenylate cyclase activity was tested. The significant adenylate cyclase activities has proved that SlyA is able to form homodimers. However, weak adenylate cyclase activities in this study has proved that MprA and PhoP are not likely to form homodimers, and no protein-protein interactions were detected in between SlyA, MprA and PhoP, which no heterodimers have formed in between three transcriptional regulators.

Antibiotic resistance in the modern era has reached near-epidemic levels, resulting in much more difficult treatment of previously well-managed pathogens. Previous understandings of how antibiotic resistance emerges failed to account for the function of the environment. Over the past 15 years, new research has provided a link between the environmental and clinical spheres of antibiotic use. This data suggests that environmental bacteria, particularly those found in livestock farming ecosystems, may significantly contribute to the overall flow of antibiotic resistance genes into human populations. The main force behind this is the utilization of antibiotics as growth promoters in animal feed supplements, seeding individual animals and their surroundings with low doses of antibiotics. Notable increases in resistance have been observed within areas that utilize these supplements, as well as in connected but unrelated systems. Waste management strategies are poorly implemented, leading to the dispersal of contaminated runoff into groundwater and riverine environments. Furthermore, existing waste processing is limited in efficacy, often releasing large amounts of unprocessed antibiotics as well as a concentrated population of resistant bacteria. Within these resistant populations, horizontal gene transfer has emerged as a vehicle for the distribution of resistance genes into other populations of bacteria. Due to the prevalence of these transfer events, a new role for the environment as a reservoir and incubator of resistance genes is proposed. Current strategies for managing the spread of antibiotic resistance are woefully inadequate, and the continued emergence of new resistance mechanisms due to negligence highlights the need for global, multidisciplinary solutions. To corral the spread of antibiotic resistance, a system is proposed that utilizes metagenomic monitoring and the enforcement of core global policies to slow the advance of resistance while waiting for novel treatment strategies to bear fruit.

Many pathogens are bacteria and antibiotic resistance is increasing. The development of novel treatments is hampered by a poor understanding of the mechanisms of their regulation. Specifically, non-coding RNAs play an important role in the internal regulation of bacteria. To further the investigation of non-coding RNA and pathogenicity, RNA sequencing data for PorX/PorY dependent regulation in P. gingivalis, a Gram negative oral pathogen was studied. The PorX/PorY two component regulatory system controls phenotypes for this bacteria's virulence including an important type IX secretion system for gingipain proteases, which degrades host cytokines, down regulating the host response by reducing inflammation. This study compared transcription of non-coding RNA in wild type and PorX knockout mutant strain, in the 33277 strain and the more virulent W83 strain in both liquid and solid cultures to identify and categorize loci of genomic sequence for further study of porX/porY regulation.

Pathogenic Gram-negative bacteria employ a variety of molecular mechanisms to combat host defenses. Two-component regulatory systems (TCR systems) are the most ubiquitous signal transduction systems which regulate many genes required for virulence and survival of bacteria. In this study, I analyzed different TCR systems in two clinically-relevant Gram-negative bacteria, i.e., oral pathogen Porphyromonas gingivalis and enterobacterial Escherichia coli. P. gingivalis is a major causative agent of periodontal disease as well as systemic illnesses, like cardiovascular disease. A microarray study found that the putative PorY-PorX TCR system controls the secretion and maturation of virulence factors, as well as loci involved in the PorSS secretion system, which secretes proteinases, i.e., gingipains, responsible for periodontal disease. Proteomic analysis (SILAC) was used to improve the microarray data, reverse-transcription PCR to verify the proteomic data, and primer extension assay to determine the promoter regions of specific PorX regulated loci. I was able to characterize multiple genetic loci regulated by this TCR system, many of which play an essential role in hemagglutination and host-cell adhesion, and likely contribute to virulence in this bacterium. Enteric Gram-negative bacteria must withstand many host defenses such as digestive enzymes, low pH, and antimicrobial peptides (AMPs). The CpxR-CpxA TCR system of E. coli has been extensively characterized and shown to be required for protection against AMPs. Most recently, this TCR system has been shown to up-regulate the rfe-rff operon which encodes genes involved in the production of enterobacterial common antigen (ECA), and confers protection against a variety of AMPs. In this study, I utilized primer extension and DNase I footprinting to determine how CpxR regulates the ECA operon. My findings suggest that CpxR modulates transcription by directly binding to the rfe promoter. Multiple genetic and biochemical approaches were used to demonstrate that specific TCR systems contribute to regulation of virulence factors and resistance to host defenses in P. gingivalis and E. coli, respectively. Understanding these genetic circuits provides insight into strategies for pathogenesis and resistance to host defenses in Gram negative bacterial pathogens. Finally, these data provide compelling potential molecular targets for therapeutics to treat P. gingivalis and E. coli infections.