Browsing by Subject "Microbiology"
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Item Biochemical and Genetic Characterization of Bacteriophage Holins(2013-11-06) To, Kam HoBacteriophages infect and kill bacterial cells. During the infection cycle, a phage attaches to the host cell surface, then ejects its DNA into the cytoplasm, where its progenies are subsequently assembled. The final step of the infection cycle is host cell lysis, which allows the progeny virions to escape into the environment. However, the timing of lysis, and thus the length of the infection cycle, is independent of endolysin biosynthesis and rather depends on the function of a second class of lysis proteins, the holins. Holins are small integral membrane proteins that accumulate harmlessly in the membrane during the infection cycle, until they suddenly form lethal lesions in the membrane at an allele-specific time. This membrane damage allows the endolysin to attack the cell wall. This dissertation focuses on several aspects of the structural and functional aspect of holins. First, Y is the putative holin gene of the paradigm coliphage P2. Although Y is not related to the S holin of phage lambda according to its primary structure, its characterization might prove useful in discerning the essential traits for holin function. In this instance, physiological and genetic approaches are utilized to show that Y exhibits the essential holin functional criteria, namely, allele-specific delayed-onset lethality and sensitivity to the energization of the membrane. These results suggest that class I holins share a set of unique features that are needed for their remarkable ability to program the end of the phage infection cycle with precise timing. Nevertheless, I report studies involving phenotypic analysis of a systematic library of clustered site-directed mutants of S105, and then conclude with experiments designed to probe the structure of the mature ?S-hole? in the membrane of the cell using chemical probes. Furthermore, I address whether the Y holin and the S21 pinholin of phage 21 effect membrane depolarization with the same all-or-nothing fashion as S while using the same tethered- cell assay previously employed for studying S. Finally, the holin and antiholin in Mu, one of the few paradigm coliphage, were identified and characterized. The introductory chapter is intended to serve as an update to the last major review on holin function in 2000.Item 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.Item 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.Item Incidence of escherichia coli O157 in feces between naturally and conventionally produced beef in Southwest Kansas(2012-05) Alexander, Clint; Miller, Markus F.; Brashears, Mindy M.; Brooks, Chance J.; Thompson, Leslie D.Changes in production methods of agricultural foods have evolved. Food and livestock are marketed in different ways including those from natural and conventional production methodologies. The objectives of this study were to evaluate the prevalence of Escherichia coli O157 between naturally and conventionally produced beef from a commercial feeding facility. Two sets of data for prevalence were obtained from two different southwest Kansas feed yards in two different trials. Positive samples from Feedlot A were evaluated for antimicrobial susceptibility. No differences (P = 0.8696) were found for prevalence of E. coli O157 between naturally- and conventionally-produced beef from samples obtained in both feedlots. A replication difference was detected (P < 0.001) with replications 1, 2 and 3 having lower prevalence of E. coli O157 as compared to replications 4 and 5. A production method by replication interaction was not detected (P = 0.3727). Feedlot A showed no difference (P = 0.5361) for prevalence of E. coli O157 with natural testing 5.11% positive and conventional testing 6.22% positive. No difference (P = 0.5747) for replication days or the production method by replication day interaction (P = 0.1608). Three of the positive isolates, all from naturally-produced cattle, from Feedlot A showed multi-drug resistance. Feedlot B showed positive results although no differences (P = 0.6568) were detected between naturally and conventionally produced beef for E. coli O157. Overall, 37.2% of the 500 samples were positive for E. coli O157:H7 with 42.4% of conventional samples and 32.0% of natural samples testing positive from Feedlot B. Sampling period did not affect (P = 0.8122) prevalence; day 1 had 38.4% positive, and 36.0% of samples tested positive on day 2. Comparing production type on sampling day, conventional positive samples dropped from day 1 to 2 (49.6% to 35.2%); however natural samples testing positive for E. coli increased from day 1 to 2 (27.2% to 36.8%). One pen of conventional samples was 100% positive. However, one pen of each treatment had 0% positive samples, both on day 2. Although no significant differences were detected between naturally and conventionally-produced beef for E. coli O157, the high percentage of positive samples for each production type as well as the multi-drug resistant isolates from naturally produced beef indicated a need for the beef and animal health industries to continue pre and post-harvest intervention methods to control E. coli O157 prevalence in the food supply to prevent food-borne illnesses.Item 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, KeithThis 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.Item 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.