When DNA damage occurs, the cell goes through various repair pathways to fix it. One result of this repair is long-tract gene conversions (LTGC). LTGCs often are present in the tumor cells of some cancers. We analyzed a collection of yeast strains containing LTGCs. Our experiments aimed to determine which DNA repair pathway was the most likely cause of each gene conversion. Using CHEF gel electrophoresis, we were able to determine the size of the repaired chromosome containing the LTGC. Next Generation Sequencing gave us the sequence of the repaired region. Using both sets of data, we were able to determine the whether the repair pathway for each gene conversion was dBIR or mmBIR. 

The behavior of multicellular communities of bacteria (i.e. biofilms) is known to significantly influence the progression of various diseases and environmental biofouling. Understanding the mechanisms by which these behaviors are regulated is essential to designing effective inhibitors. The soil bacteria, Myxococcus xanthus, is an excellent model system for examining multicellular behavior in bacteria. M. xanthus can enter a developmental program during which cells differentiate into spatially distinct cell fates: 1) spore filled fruiting bodies, 2) programmed cell death, or 3) a persistor-like state. MrpC, a CRP/Fnr family transcription factor, is a global regulator of multicellular development and required for coordinated cell fate segregation. MrpC is subject to numerous levels of regulation, both transcriptional and post-transcriptional, which leads to differential accumulation of MrpC in the different cell types. To determine how MrpC accumulation is differentially regulated, we set out to understand how mrpC transcription was regulated. Using fluorescent transcriptional reporters and electrophoretic mobility shift assays, we determined that MrpC functions as a negative autoregulator, interfering in activation of mrpC expression. We then set out to determine the role of MrpC autoregulation in development by driving mrpC expression from a promoter in which autoregulation had been perturbed. Using a high-resolution developmental assay, we identified that perturbing MrpC autoregulation resulted in asynchronous and uncoordinated development. Data from fluorescent reporters for mrpC expression suggest that MrpC autoregulation plays a role in coordinating development by limiting cell-to-cell variability in gene expression.

With the rise of infections caused by multidrug resistant bacteria, there is a need for new antibiotics. We have identified many soil bacteria that produce antibiotics effective against carbapenem-resistant Enterobacteriaceae (CRE) grown in microcosm environments. We can now recreate these effects in simple environments by adding different simple or complex polysaccharides to our media and co-culturing these antibiotic-producing strains with Escherichia coli and Klebsiella pneumoniae. The addition of mannitol or potato starch were most effective at inducing this activity. Mass spectrometry and NMR will be used to identify the chemical components responsible for the antimicrobial activity.

Antibiotic-resistant Staphylococcus aureus is the leading cause of healthcare-associated infections. An expansive tissue tropism and ability to withstand antimicrobial therapies make S. aureus a significant clinical challenge. S. aureus proliferates in diverse host tissues using a versatile metabolism that fluctuates between aerobic respiration and fermentation. Aerobic respiration represents another layer of metabolic flexibility as a branched respiratory chain composed of two terminal oxidases, QoxABCD and CydAB, catalyze the terminal reduction of oxygen to water. CydAB and QoxABCD are required to colonize the heart and liver, respectively, underscoring the importance of aerobic respiration to pathogenesis. These facts support the hypothesis that the terminal oxidases have unique biophysical properties that allow them to function in distinct environments. To define these properties, we seek to identity host-derived factors that influence the function of each terminal oxidase, filling considerable gaps in our knowledge of staphylococcal pathogenesis. In this study, we found that genes within the exogenous fatty acid incorporation pathway are significantly downregulated in a qoxA mutant compared to wildtype. Exogenous fatty acid incorporation is utilized by S. aureus to synthesize phospholipids and has been proposed to play a role in detoxifying the membrane from harmful concentrations of free fatty acids. We observe that saturated fatty acids, which are innocuous to aerobically respiring cells, severely limit the proliferation of respiration-arrested cells. Our data and the fact that the liver is a significant host reservoir of free fatty acids, support the hypothesis that host-derived fatty acids represent an extrinsic challenge for respiration-impaired S. aureus during infection. Specifically, we reason that the aberrant accumulation of free fatty acids leads to toxicity in metabolically-restricted S. aureus. This research is the first to link antimicrobial activities of fatty acids to the metabolic status of a bacterial pathogen and establishes respiration as a fatty acid-resistance determinant. 

Dental caries, also referred to as tooth decay or cavities, is caused by erosion of enamel on the tooth surface and without intervention can eventually lead to tooth loss. One of the main factors leading to caries is the accumulation of bacterial biofilm, also called plaque. The gram positive bacterium, Streptococcus mutans, is highly correlated with the initiation of dental caries due to its ability to produce a sticky matrix of extracellular polysaccharides (EPS) while growing in the presence of sugars. Additionally, S. mutans can metabolize dietary sugars results in the production of lactic acid which can directly degrade enamel and lead to tooth decay. Four serotypes of S. mutans exist based on extracellular characteristics: c, e, f, and k. Almost 80% of S. mutans found in the oral cavity are serotype c, while serotypes e and f are not as prevalent, yet still contribute towards dental disease. Moreover, serotype k is commonly associated with cardiovascular disease. The objective of this study is to determine if serotype influences the ability of S. mutans to form biofilm in vitro. Additionally, the CRISPR-Cas system of bacterial defense against foreign DNA elements has recently been shown to correlate with increased virulence in S. mutans. Therefore, we propose to determine the prevalence of CRISPR arrays in different serotypes of S. mutans and the correlation with biofilm formation. These studies will enhance our understanding of the impact of S. mutans serotype and CRISPR presence on disease-causing ability persistence/prevalence in the population, as well as the possible resistance to foreign genetic elements.

Common barriers to productively facilitating scientific research with a full-time undergraduate student may include lack of time for the student to consistently conduct research and variations in scientific principle comprehension. Therefore, maximizing the efficiency of laboratory training is essential for the process of developing a student’s ability to conduct benchtop science while learning to effectively and critically analyze a research project. The success of the training period is of high importance to foster a mutually beneficial partnership between faculty and student. The goal of this project was to have an experienced undergraduate lab member develop a series of regimented experiments specifically designed to efficiently train new undergraduate research students in the foundational principles and techniques of molecular biology regardless of prior lab experience. The project focused on determining the most efficient sequence of instruction and experimentation. The teaching design was based on one fundamental molecular biology experiment: Polymerase Chain Reaction (PCR). Using this technique as the basis of introduction into the research setting, the new undergraduate members were taught how to appropriately write in a lab notebook, make solutions, learn equations and unit conversions, all while being trained in the use of basic lab equipment. Through this process we aspired to determine the most appropriate sequence of instruction, experimental techniques, and reinforcement needed for an incoming student to successfully investigate research questions, regardless of their previous research experience or year of study in an undergraduate program.

Infected cell protein 0 (ICP0) is one of the immediate early proteins of herpes simplex virus-1 (HSV-1) that is mainly responsible for counteracting host antiviral defense mechanisms.  Upon HSV-1 infection, several cellular antiviral factors converge at the incoming viral genome, attempting to silence viral genes. Simultaneously, ICP0, which is an E3 ubiquitin ligase, targets some of these host restrictive factors for proteasomal degradation to alleviate host defense and enhance viral gene expression. Promyelocytic leukemia protein (PML) is an important cellular protein involved in several cellular pathways including antiviral defense that gets degraded by ICP0 in this process. Previously, we reported that ICP0 degrades PML isoforms differentially and a bipartite domain located upstream and downstream of the RING-type E3 ubiquitin ligase domain of ICP0 mediates its interaction with PML I. In the current study, we report that (i) either one arm of the ICP0 bipartite PML I-interacting domain is sufficient for PML I interaction, ubiquitination and degradation, whereas deletion of both arms of the bipartite domain hinders binding, ubiquitination and consequently the degradation of PMLI. (ii) The two arms of bipartite PML I-interacting domain in ICP0 share sequence similarity, which is necessary and sufficient for PML I interaction and ubiquitination. (iii) The homologous sequences in the two arms of bipartite PML I-interacting domain also share sequence similarity to the C-terminus of PML I. (iv) Deletion of PML I C-terminus inhibits the ICP0-PML I interaction. These results suggest that ICP0 may have evolved to partially mimic PML I sequence. This possible adaptation enables ICP0 to target PMLI specifically and regulate its ubiquitin-mediated degradation.

Candida albicans is an opportunistic fugal pathogen and a member of the normal human microbiota. Various environmental conditions stimulate changes in cellular morphology between round yeast cells and filamentous hyphae and pseudohyphae. One such stimulus is growth embedded in solid media. Examination of scientific literature revealed that several mutant strains known to grow filamentously under yeast conditions had not been tested in embedded growth. One example is a strain over-expressing the transcription factor SFL2. Our initial goal was to close this gap in our knowledge of these strains. Interestingly, our experiments revealed that over-expression of SFL2 was also able to overcome the repression of filament induction that results from over-expression of the repressor NRG1. This effect appears to be specific to embedded conditions, since in several liquid media tested simultaneous over-expression of SFL2 and NRG1 produces only elongated yeast cells. We are continuing to examine this question by growing our over-expression strain in other conditions known to induce filamentous growth.

Salmonella enterica serovar Typhimurium is a Gram-negative pathogen responsible for a large percentage of food-borne illnesses in the US each year. Upon entry, S. Typhimurium invades the epithelial cells and Peyer’s patches of the small intestine via a type III secretion system. Invasion requires a region of the genome known as Salmonella Pathogenicity Island 1 (SPI-1), and intracellular survival requiring Salmonella Pathogenicity Island 2 (SPI-2). Central to the upregulation of SPI-1 is the gene hilA, which is both positively and negatively regulated via a series of proteins that senses the bacteria’s current environment.  One of the major negative regulators of hilA is the gene hilE. Previously, it was found that the genomic area surrounding hilE carries key characteristics commonly seen in pathogenicity islands. The purpose of this research is to characterize the effects two polar mutations, Δ4501 and Δ4502, located in this region, have on virulence as part of a continuing study of this pathogenicity-island-like region. Current work was done by measuring hilA expression under inducing and non-inducing conditions via beta-galactosidase assays in mutant and wild type strains. Further, motility and twitching assays were performed on both mutant and wild type cells. Results indicate no significant change in expression of hilA in the absence of Δ4501 and Δ4502 in both inducing and non-inducing conditions. Results from the twitching and motility assays are currently in progress. Thus far, the results indicate that Δ4501 and Δ4502 have insignificant effects on S. Typhimurium virulence. Future work will focus on the effects Δ4501 and Δ4502 have on invasion through invasion assays, the characterization of expression of other known regulators, macrophage survival and adherence.

At the nanoscale, cellular pathways are frequently organized upon scaffolding complexes that physically co-localize related components. Natural Nano-scaffolds act to increase pathway flux and fidelity, while also insulating the pathway from unwanted side-reactions and reducing accumulation of pathway intermediates. An outstanding goal for biological engineers is to recapitulate the advantages of natural scaffolds to improve the performance of heterologously installed pathways. Our approach is to use a naturally-occurring protein that self-assembles into defined intracellular structures (bacterial microcompartment shell proteins) as “building blocks” for the assembly of new, user-defined Nano-structures. Here, we show that heterologous expression of these self-assembling building blocks can be used to form macromolecular protein assemblies both in vitro and in vivo. We describe our efforts to attach functionalizing adaptor domains to these protein building blocks in order to allow for programmable protein-protein interactions on the surface of these Nano-scaffolds. Within the context of this broader goal, my project seeks to understand how attaching adaptor domains influences the self-assembly characteristics of shell proteins under a range of different physiological temperatures and pH ranges. By analyzing their ability to continue to form higher order structures, even with the addition of these functional adaptor domains, we aim to characterize a promising biomaterial that could be used as a tool in future bioengineering purposes.

The fungus Candida albicans is in most human intestines and mucous membranes, and it typically does not cause disease in healthy individuals. However, with changes in the host, such as in immune compromised individuals, disease can arise due to C. albicans ability to change cellular shape between round yeast and elongated filaments. In C. albicans cellular shape is regulated by a genetic program. The function of many genes associated with filamentation is unknown. Our goal is to understand the particular contribution to this process of one gene, FGR16. FGR16 overexpression caused increased biofilm growth in some media, and increased clumping in some broth cultures. Changes in plate growth included reduced filamentous growth, as well as increased invasion.  These results confirm that FGR16 overexpression affects cell shape. We are testing how well this gene’s function is maintained between organisms; by overexpressing homologs in C. albicans and looking for effects on cell shape.

Lactobacillus rhamnosus GG (LGG) is one of the best studied probiotic organisms. The ability of probiotics to adhere to other microorganisms and the intestinal epithelium is thought to play a major role in their protective functions. Coaggregation is thought to be an important mechanism for biofilm formation by microorganisms. Recent studies from our lab suggest that there is extensive coaggregation between human gut microbes. It is hypothesized that nutritional variation may affect the ability of LGG to coaggregate and form biofilms, and thus affect its probiotic characteristics and ability to colonize the gastrointestinal tract. The goals of this study were to examine the ways in which nutrient variation affects coaggregation between LGG and other intestinal species; biofilm formation between LGG and B. thetaiotaomicron wild-type or B. thetaiotaomicron capsule-deficient mutant in vitro; and LGG cell-surface properties and structures. Lactobacillus rhamnosus GG was cultured anaerobically in different formulations of tryptone, yeast extract, and glucose (TYG) medium in order to simulate nutritional shifts Coaggregation ability between LGG and 22 Bacteroides spp. and Parabacteroides spp. and 8 mutant capsular types of Bacteroides thetaiotaomicron were tested. There was a significant difference (p<0.001) in coaggregation when compared by media type. Twenty-four-hour static biofilm assays were conducted in plastic 24-well microplates, and the optical density of the stained biofilms was determined in order to quantify the level of adherence. Biofilm formation of LGG with either the B. thetaiotaomicron wild-type strain or the B. thetaiotaomicron capsule-deficient mutant strain was observed to significantly differ by media type (p<0.001). Hydrophobicity values of LGG grown under differing nutrient conditions were also examined, and were found to significantly differ by media type (p<0.001). These results suggest that different nutrient conditions may enhance the ability of LGG to act as a successful probiotic.

Orthogonal regulation is important for the field of synthetic biology in order to reduce cross-talk between endogenous and recombinant regulators in engineered microbes. My goal for this project was to determine the feasibility of using a carbohydrate sensing regulatory system from the thermophilic bacterium Clostridium thermocellum in Bacillus subtilis. Both Firmicutes evolved from a common ancestor; however, this regulatory system evolved in C. thermocellum and not in B. subtilis, making it a candidate for orthogonal regulation. If successful, this system will be used to construct a biosensor microorganism, using B. subtilis, that will respond to plant-derived carbohydrates such as cellulose, xylan, and pectin, by producing green fluorescent protein. C. thermocellum anti-sigma and sigma factors will be cloned into a replicating B. subtilis plasmid using isothermal assembly, while the cognate promoter will be cloned in front of gfp on a non-replicating plasmid using Golden Gate assembly. We modified an existing non-replicating plasmid for use in Golden Gate cloning, including replacing the strong lactose-inducible promoter with a lacZ cassette flanked by BsaI sites and using site directed mutagenesis to remove a BsaI recognition site from the β-lactamase gene. Both plasmids were transformed into supercompetent B. subtilis SCK6. Using a spectrofluorimeter, we quantified levels of non-target expression from the C. thermocellum promoter in B. subtilis. Constructed plasmids and engineered B. subtilis strains will ultimately be used to construct biosensors for use in directed evolution of carbohydrate recognizing proteins.

Pseudomonas aeruginosa is an antibiotic resistant bacterium that is known to cause infections of blood. This study examined growth of P. aeruginosa ATCC 13388 with a phage isolated from stream sediment in presence of blood and blood serum. An antibiogram made on Luria-Bertani (LB) agar revealed that the host strain of P. aeruginosa was able to grow up to the edge of 3 out of 5 different antibiotic discs. The Enterotube II/EnteroPluri-Test, which is a multiple biochemical test system made of 12 test chambers in a series, indicated that the host strain when inoculated in presence or absence of phage was nonfermentative. With prolonged incubation positive reactions for arabinose or glucose were occasionally produced. In the routine test dilution (RTD) assay, to discover the highest titer of phage to give complete lysis, carried out on trypticase soy agar (TSA) with sheep blood, clear zones on lawns of cells on agar for dilutions up through 10 raised to the power of -6 were apparent. This RTD result was the same as observed for the phage with the assay performed on brain heart infusion (BHI) agar without blood. The effect of phage on growth of P. aeruginosa in blood serum was assessed by measurement of culture optical density (OD) with a Klett-Summerson colorimeter. Inclusion of phage at a multiplicity of infection (MOI) of approximately 1.0 resulted in a bacterial culture with OD 35, in contrast to a culture without phage with OD 160 after 6 days. In BHI, in absence of blood factors at an MOI of approximately 100, a culture containing phage with OD 38 in contrast to a culture without phage with OD 138 resulted in 24 h. However, when an MOI of approximately .01 was used the culture with phage was only slightly inhibited. Streak plate cultures after experiments produced survivor colonies that were green from pyocyanin pigment. Development of bacterial biofilm on blood was inhibited by the phage. Growth of the bacteria in blood serum was slow and phage further decreased growth.