Browsing by Subject "Microbiology"

Now showing 1 - 12 of 12

Applications of large, heterogeneous datasets in understanding and treating pathogenic microbes
(2021-01-22) DuPai, Cory David; Wilke, C. (Claus); Davies, Bryan William; Maynard, Jennifer A.; Press, William H.; Barrick, Jeffrey E.
Major advances in a myriad of technologies over the past two decades have led to a remarkable increase in the generation of biological data. In response to this increase, researchers have developed methods to pool and analyze large, heterogeneous datasets for novel insights. Here I do just that, leveraging existing data to expand our understanding of therapeutic proteins and pathogenic microbes. In Chapter 2 I outline major shortcomings in existing viral annotation standards using metadata from all influenza A sequences submitted to the GISAID database between 2005 and 2018. I further establish updated nomenclature standards to improve annotation accuracy moving forward. In Chapter 3 I use published Vibrio cholerae sequencing data to derive a comprehensive gene coexpression network. This network provides direct insights into genes influencing pathogenicity, metabolism, and transcriptional regulation, further clarifies results from previous sequencing experiments in V. cholerae, and expands upon micro-array based findings in related gram-negative bacteria. In Chapter 4 I systematically probe all 49,000 unique beta hairpin substructures contained within the Protein Data Bank to uncover key characteristics correlated with stable beta hairpin structure, including amino acid biases and enriched inter-strand contacts. I also establish a set of broad design principles that can be applied to the generation of libraries encoding bioactive proteins. These findings highlight the untapped potential, promise, and power of pooled analyses using large, heterogeneous datasets
Bacterial pathogen adaptation during human infections
(2018-06-25) Crofts, Alexander; Davies, Bryan William; Trent, Michael Stephen; Payne, Shelley M; Ochman, Howard; Croyle, Maria A
Like all organisms on earth, bacteria must adapt to changes in their environment to survive. Thus, discovering bacterial adaptations reveals the tools bacteria use to be successful. Identifying how pathogenic bacteria adapt during infections can consequently identify the tools bacteria use to cause disease, and therapy design can then consider inhibiting these tools to treat or prevent infections. Here, the ways in which two worldwide human intestinal pathogens, Campylobacter jejuni and Enterotoxigenic E. coli (ETEC), adapt to the human host during infections are explored. Bacteria were studied directly in infected samples from controlled human infection models. In C. jejuni, genetic adaptations that were selected for during acute and persistent human infections identified the role of a previously uncharacterized flagellar modification gene during persistence. In ETEC, the bacteria’s ability to sense oxygen was linked to global virulence gene expression in human infection samples as well as biofilm formation. As environmental ETEC biofilms are associated with seasonal ETEC epidemics, oxygen sensing likely contributes to human infection inside and outside of the host. Together, these data demonstrate the scope of pathogen adaptation during infections, identified new targetable virulence factors, and can thus aid the design of new therapies
Comparative systemic analysis of human immunoglobulin repertoires
(2019-01-31) King, Gregory Ryan; Georgiou, George
The humoral immune system is majorly composed of B cells producing effector immunoglobulin molecules, the vast diversity of which allow for the neutralization of pathogenic threats never previously seen by the immune system. High-throughput sequencing technology has allowed this vast repertoire to be characterized and quantified, but understanding this complex system requires methods of comparison to identify and differentiate B cell populations. In this thesis, differences between groups of repertoires within individuals are analyzed at both the cellular and proteomic level. Novel experimental techniques and visualization methods will allow for the analyses of several such high-dimensional complex systems, leading to a fuller picture of the B cells’ contribution to the immune system.
Creation of a viable csrA mutant in Vibrio cholerae
(2013-08) Thomas, Martha Barnett; Payne, Shelley M.
Vibrio cholerae, the causative agent of cholera, has been a lethal enteric pathogen to humans for most of recorded history. Even though it is well studied, it still kills many people every year due to rapid and severe dehydrations from diarrhea. Part of what makes V. cholerae such an effective pathogen is its ability to control virulence factors depending on its environment. ToxR is a major virulence protein that has upstream control of most of the virulence genes that are turned on when in a human host. Two of the most critical virulence factors, toxin coregulated pilus and cholera toxin are controlled by ToxR. CsrA is a protein that regulates many cellular functions in V. cholerae, including glycogen synthesis, motility, and biofilm production. Preliminary data suggests a link between CsrA and the regulation of ToxR. In order to study CsrA as it relates to ToxR regulation, a csrA mutant must be generated in V. cholerae. CsrA plays such an important role in glycogen metabolism that a csrA mutant is not viable due to excessive glycogen levels. In order to make a viable csrA mutant, glycogen synthesis has to be turned off. In this research, I attempt to make a viable V. cholerae csrA mutant by deleting csrA in a strain that is deficient for glycogen synthesis (glg). Normally without CsrA, glycogen in the cell would increase to a detrimental level. Since a glg⁻ csrA⁻ mutant lacks the ability to make glycogen, the levels never reach a lethal level, allowing the mutant to survive without functional CsrA. Such a glg- csrA- double mutant's ToxR regulation can be studied by growth in various media by measuring OmpU and OmpT expression. Using PCR, restriction enzymes, and DNA ligase, a suicide plasmid was created containing sequences that flank the csrA gene but instead of the csrA gene, a chloramphenicol resistance cassette was inserted. Through bacterial conjugation this plasmid was introduced into three V. cholerae glg- strains. Allelic exchange was carried out utilizing the homology between the DNA flanking wild type csrA and the csrA deletion with chloramphenicol cassette. This first crossover event was initiated with the requirement of the [pi] protein for the plasmid to replicate. Without the pir gene to create [pi] protein, selection for antibiotic resistance required that the plasmid integrate into the genome. This was selected based on the plasmid encoded ampicillin resistance. After the second crossover event, there were two possible outcomes of excision: reverting to wild type csrA or retention of the csrA mutation. The csrA mutant was selected based on its sucrose and chloramphenicol resistance and ampicillin sensitivity.
Development of new strategies to map and regulate large RNA regulatory networks
(2016-10-11) Sowa, Steven William; Contreras, Lydia M.; Baldea, Michael; Barrick, Jeffrey; Iyer, Vishwanath; Harshey, Rasika
Global regulators are critical controllers of cellular function. They possess the ability to coordinate multiple cellular pathways simultaneously to orchestrate a unified response to environmental change. Recent research has demonstrated that both proteins and RNAs can function in key regulatory roles and each provides unique advantages in their control characteristics. Given the power of these regulators, there is strong interest in utilizing regulatory systems in a variety of metabolic engineering applications including coordination of metabolic pathways and as dynamic pathway controllers. However, the potential to utilize these systems to produce dynamic and coordinated metabolic responses is only beginning to be realized. My dissertation focuses on characterizing global regulatory systems and specifically small RNA (sRNA) based regulatory systems for their use as dynamic global controllers for metabolic engineering. Chapter 2 starts by discussing a computational analysis of how a group of genes dynamically controlled by a single regulator can produce higher metabolite levels over time. This Chapter demonstrates dynamic control as an underutilized strategy to improve metabolite production. Chapter 3 evaluates how to characterize large regulatory systems using the E. coli Carbon Storage Regulator as a case study. It interweaves multiple lines of omics evidence and follow up experiments to determine the regulatory targets of the Csr system. Using these data, we constructed a thermodynamic model to predict if CsrA will cause repression of cellular genes (Chapter 4). The work in these chapters represents a new way of thinking about sRNA regulators and their role in metabolic engineering and has broad applicability to other Protein-RNA regulators and other regulatory elements.
Engineering the gut microbiome of honey bees
(2020-06-22) Leonard, Sean Patrick; Moran, Nancy A., Ph. D.; Barrick, Jeffrey E.; Ochman, Howard; Marcotte, Edward; Davies, Bryan
Honey bees are critically important commercial pollinators and model systems for insect physiology and behavior. Honey bees are also suffering dramatic declines worldwide due to many factors, including agricultural practices, parasites, and pesticide use. These bees house a simple, conserved gut microbiome that is important for their health. Can we use this gut microbiome to protect bees in new ways? Synthetic biology combines recombinant DNA technology and rational design principles to redesign biological processes. Microbiome engineering applies synthetic biology and engineering principles to microbial communities to improve or expand their functions. Because of their agricultural importance, history as a model organism, and simple gut microbiome, honey bees are a promising testbed for the nascent field of microbiome engineering. In Chapter 1 I provide a brief introduction to the host-associated microbiomes, honey bees, and synthetic biology. In Chapter 2, I develop broad-host-range tools for genetic manipulation of bacteria from honey bees and show that genetically engineered bacteria can recolonize and function in bees. This lays the groundwork for follow-on efforts to both study and further engineer the bee gut microbiome. In Chapter 3, I describe the application of these genetic tools to engineer core microbiome member Snodgrassella alvi to produce double-stranded RNA (dsRNA) and thereby induce RNA-interference (RNAi) in bees. Activating RNAi enables bee researchers to study specific bee genes. In the future this technique may be used to protect honey bee hives from viruses and parasitic mites. In Chapter 4, I describe a computational approach for designing and evaluating defined bacterial communities and discuss using these defined communities in honey bees. These chapters together demonstrate how the bacterial community native to an organism can be modified and address several technical limitations of microbiome engineering in honey bees. Finally, I discuss the next steps for continuing this work.
Evolution of microbial populations with spatial and environmental structure
(2010-05) Miller, Eric Louis; Meyers, Lauren Ancel; Bennett, Philip C.; Bull, James J.; Hawkes, Christine V.; Hillis, David M.
Rarely are natural conditions constant, but generally biologists study microbes in artificially constant environments in the laboratory. I relaxed these assumptions of constant environments through time and space as I investigated how microbial populations evolve. First, I examined how bacteriophage evolved in the presence of permissive and nonpermissive hosts. I found that bacteriophage evolved discrimina- tion in mixed environments as well as in one of two environments with homogeneous, permissive hosts. This showed the asymmetry of host-shifting in viruses as well as the possibility of large, and somewhat unpredictable, pleiotropic effects. Secondly, I reconstructed ancestral environmental conditions for soil bacteria groups using phy- logenetics and environmental variables of extant species’ habitats. These generaliza- tions suggested characteristic phenotypes for several phylogenetic groups, including uncultured Acidobacteria. Lastly, I collected genetic sequences and global collection information for 65 bacteria genera across the domain. In examining the relation- ship between genetic distance, environmental conditions, and geography, I observed positive relationships specifically between genetic distance and geography or genetic distance and environmental conditions for bacteria from land sites but not from wa- ter sites. Phylogenic classifications or phenotypes of the genera could not predict these correlations. In all of these projects, variations in the environment created evolutionary signals that hinted at past environments of microbial populations.
Investigating sociomicrobiology by integrating micro 3D printing with quantitative analytical techniques
(2019-02-05) Fitzpatrick, Mignon Denise; Shear, Jason B.; Brodbelt, Jennifer; Anslyn, Eric; Roberts, Sean; Hoffman, David
Antibiotic resistant polymicrobial infections have become a source of great concern in recent years both in clinical settings as well as in basic and medical research. Incidence of resistance and increased virulence, which typically emerge within small, dense cellular ensembles on picoliter scales, is on the rise and scientists are just beginning to understand the complexity of these dangerous bacterial populations. To that end, the research in this dissertation has sought to analyze the complex social interactions of micro 3D (µ3D) printed bacterial colonies with a variety of analytical techniques. Through characterization of the µ3D printed hydrogels themselves, and by pairing this technology with fluorescence and confocal microscopy, electrochemical studies, and mass spectrometry, important insights regarding the sociomicrobiology of these bacterial communities emerge. The Shear lab has previously employed µ3D printing of bacterial aggregates to study microbial populations in environments that reproduce attributes associated with complex spatiotemporal in vivo conditions to a much greater extent than traditional culture techniques. Combining this technology with advanced imaging approaches has enabled a detailed investigation into properties of intra- and inter-species cooperation, including factors that influence antibiotic resistance and virulence. The goal of the work presented here is to integrate quantitative and qualitative analytical techniques with µ3D printing technology to enable novel approaches for studying interactions, both within and between small bacterial aggregates in complex microbial environments. This information will be vital in the next steps toward designing better and more efficient strategies for combating complex pathogenic communities that exist within polymicrobial infection environments.
Learning through interaction and embodied practice in a scientific laboratory
(2012-05) Mey, Inger Hansen, 1941-; Keating, Elizabeth Lillian; Sherzer, Joel; Streeck, Jürgen; Hartigan, John; Walters, Keith
This study purports to explore how apprentices in microbiology, through interaction and multimodal activities, acquire the knowledge and skills that are necessary for doing scientific experiments. It aims to examine the ways novices learn to scrutinize and discuss the data under investigation, how experts communicate scientific knowledge about microbes to novices, and how experts and novices together create new scientific knowledge during the apprenticeship. Furthermore, this study aims at explaining the various ways narratives contribute to the socialization of the apprentice into the workplace and the scientific field, and how stories help retain knowledge, gained in one situation, to be used in other contexts and situations. To achieve this aim, I videotaped daily activities in a small microbiology lab, focusing on detailed observations of experts and novices as they engaged in teaching and learning. I was especially interested in what kinds of innovative symbolic communication resources would be invoked during such educational activities. In addition, I collected data pertaining to how the apprentice was socialized into this particular community of practice. I applied a ‘situated learning’ approach to the analysis of the instructional data, as well as discourse analytic and social semiotic methods of analyzing verbal and nonverbal, embodied interaction. I found that researchers, by using embodied and semiotic resources, created moments of shared participation between themselves and their scientific objects. Likewise I found that gestures shaped objects and concepts, and brought these into an intersubjective space where researchers, tools, instruments, and concepts interacted in a collaborative architecture. I named the specific literacy prevalent in scientific experimentation (reading and understanding graphs, diagrams, pictures, etc.) as ‘science literacy’, to distinguish it from the term ‘scientific literacy’, a general understanding of popularized scientific topics. Blurred boundaries were discovered between the living organisms and their semiotic representations whenever the expert and the novice referred to the living organisms in their discussions concerning graphs and diagrams. The researchers changed their terminology, depending on the bacteria changing from animate to inanimate status. Finally, I discovered the significance of contextual tellability in narratives functioning both as introduction to the workplace and as memory devices.
New tools and applications for genetically engineered insect symbionts
(2022-08-04) Elston, Katherine Marie; Barrick, Jeffrey E.; Moran, Nancy A; Davies, Bryan W; Havird, Justin C
Insects play a broad range of roles in natural ecosystems. They pollinate plants, recycle resources, and spread diseases. One integral component of the biology of many of these insects is the symbiotic bacteria that live inside of them. The relationships between symbionts and their insect hosts are well-studied in model systems like aphids, but despite this work, the ability to study genetic components of these relationships has been lacking. A major factor in this limitation is the deficit of tools that are available for genetically manipulating both non-model bacteria and insect species. In this work, I examine these limitations, along with the possibilities for the study of insect biology and control of insect pests that may arise if we can overcome them (Chapter 1). I develop methods to engineer recently isolated symbionts of aphids (Chapter 2), and fruit flies (Chapter 3). I then adapt these engineered aphid symbionts to try to alter aphid gene expression through symbiont-mediated RNAi (Chapter 4). I conclude with a summary of our results and the implications this work may have for the future of insect symbiont engineering (Chapter 5)
Physical characterization of bacterial biofilm polymer networks to determine the role of mechanics in infection and treatment
(2018-11-29) Kovach, Kristin N.; Gordon, Vernita Diane; Florin, Ernst-Ludwig; Marder, Michael P; Smyth, Hugh D; Lynd, Nathaniel
Biofilms are communities of microorganisms that produce a matrix of extracellular polymers to surround and protect themselves from external forces in their environment. This communal lifestyle is incredibly beneficial for microorganism survival. Characterization of the mechanical properties of biofilms is a vital and understudied component of fully understanding these biological systems. In this dissertation, we break down the mechanical response of the Pseudomonas aeruginosa biofilm by its constituent polymers. These bacteria produce unique polymers to resist a variety of stresses. In the first part of this dissertation, using oscillatory bulk rheology, we characterize the viscoelasticity of biofilm polymer networks. Using genetically manipulated lab strains of P. aeruginosa, we isolate the mechanical response of each polymer by analyzing biofilms comprised primarily of one type of polymer. We find that the polymers have unique mechanical properties: some increase the yield strain and others increase elastic modulus. In strains of P. aeruginosa isolated from chronic infections, we find that the bacteria evolve to increase production of polymers that maximize the energy required to yield the matrix. In the second part of this dissertation, we work to mechanically compromise each of the polymers in the matrix. By attacking different matrix components, we learn more about the structural properties that give rise to mechanical properties as well as identify the most promising therapeutic treatments to break down biofilm infections. We find that specific enzymes are useful for decreasing yield strain of biofilms and increasing the diffusivity of the matrix. Decrease in yield strain means that biofilms will take less deformation before losing mechanical integrity, and the increase in matrix diffusivity means that current treatments such as antibiotics are more effective as the antibiotics can more easily reach the bacteria in the matrix to effectively kill them. This dissertation treats biofilms as polymer networks, divorcing the analysis from biological responses, in an attempt to well-characterize the understudied mechanical properties of biofilms. By approaching these systems from a physical standpoint, we are able to learn more about biofilms by breaking the mechanical response into constituent components, as well as learn about how enzymatic treatments alter biofilm properties.
Studies in bacterial genome engineering and its applications
(2014-05) Enyeart, Peter James; Ellington, Andrew D.
Many different approaches exist for engineering bacterial genomes. The most common current methods include transposons for random mutagenesis, recombineering for specific modifications in Escherichia coli, and targetrons for targeted knock-outs. Site-specific recombinases, which can catalyze a variety of large modifications at high efficiency, have been relatively underutilized in bacteria. Employing these technologies in combination could significantly expand and empower the toolkit available for modifying bacteria. Targetrons can be adapted to carry functional genetic elements to defined genomic loci. For instance, we re-engineered targetrons to deliver lox sites, the recognition target of the site-specific recombinase, Cre. We used this system on the E. coli genome to delete over 100 kilobases, invert over 1 megabase, insert a 12-kilobase polyketide-synthase operon, and translocate a 100 kilobase section to another site over 1 megabase away. We further used it to delete a 15-kilobase pathogenicity island from Staphylococcus aureus, catalyze an inversion of over 1 megabase in Bacillus subtilis, and simultaneously deliver nine lox sites to the genome of Shewanella oneidensis. This represents a powerful, versatile, and broad-host-range solution for bacterial genome engineering. We also placed lox sites on mariner transposons, which we leveraged to create libraries of millions of strains harboring rearranged genomes. The resulting data represents the most thorough search of the space of potential genomic rearrangements to date. While simple insertions were often most adaptive, the most successful modification found was an inversion that significantly improved fitness in minimal media. This approach could be pushed further to examine swapping or cutting and pasting regions of the genome, as well. As potential applications, we present work towards implementing and optimizing extracellular electron transfer in E. coli, as well as mathematical models of bacteria engineered to adhere to the principles of the economic concept of comparative advantage, which indicate that the approach is feasible, and furthermore indicate that economic cooperation is favored under more adverse conditions. Extracellular electron transfer has applications in bioenergy and biomechanical interfaces, while synthetic microbial economics has applications in designing consortia-based industrial bioprocesses. The genomic engineering methods presented above could be used to implement and optimize these systems.