Our lab created ionizing radiation (IR) resistant populations of E. coli MG1655 through twenty cycles of exposure to IR and outgrowth. To identify the genetic alterations responsible for increased IR tolerance, a total of seven isolates from the evolved final population were re-sequenced. However, the individual isolates sequence analysis revealed wide variety of unique genomic alterations with little overlap between the seven isolates and therefore, did not reveal the clear picture of the mutations responsible for IR resistance. Our current research focused on distinguishing the relevant mutations from the non-relevant mutations that contribute to IR resistance by using two methods: population sequencing and TaqMAMA with the assumption that beneficial mutations evolve and accumulate in the final population. A 255X deep coverage sequencing of the genomic DNA from the IR resistant final population determined mutant allelic percentage in this population for each polymorphism detected. TaqMAMA assay acted as a tool for verifying the sequencing results. This assay also revealed two evolutionary phenomena: clonal interference and hitchhiking suggesting that not all polymorphisms seen in the final population could contribute to IR resistance. The analysis of population sequencing results along with the phylogenetic analysis reduced the number of potential candidates responsible of IR resistance in these isolates. These possible contributors enable at least three unique mechanisms in the seven isolates and the survival data revealed that the related isolates have same levels of IR resistance possibly from the same mechanism. The identified candidates were transferred into the parent strain alone or in combination and the resulting strains were tested for survivability against IR. This experiment identified the possible reasons of IR resistance in these isolates. In fact, a RecA modification has been determined to be a lone contributor for IR resistance in the four isolates. The methods used here, enabled us to separate relevant mutations that accumulated during evolution from non-relevant mutations that do not and thus helped to reduce the number of alleles possibly that could contribute to IR resistance. However, genetic characterization is required to confirm their role in IR resistance due to complexity that exists in allelic selection during evolution.

The DedA protein family is a highly conserved family of membrane proteins, with members present throughout all domains of life: Bacteria, Archaea, and Eukarya. Investigation of the DedA membrane protein family began with the isolation of BC202, an Escherichia coli mutant with in-frame deletions of two DedA proteins of unknown function (YqjA and YghB) that share 61% amino acid identity. BC202 demonstrates temperature sensitivity, inefficient cell division, an altered phospholipid composition, increased expression of extracytoplasmic stress response pathways, as well as an inability to maintain the cell membrane proton motive force (PMF). Additionally, Borrelia burgdorferi has a single DedA homolog (Bb0250), deletion of this gene was only possible in the presence of a cloned and inducible copy of the gene. The essentiality of the DedA membrane protein family in B. burgdorferi, instigated the investigation into the essentiality of the family in E. coli, which has eight individually non-essential DedA genes. Discussed herein is the generation of mutants in which all eight E. coli DedA genes are deleted, which again, was only possible in the presence of a cloned and arabinose inducible DedA protein. One mutant was further characterized, BAL801 (cloned EcDedA), identifying that the essential function of the E. coli DedA membrane protein family may be to play a vital role in the proper maintenance and segregation of the bacterial nucleoid. Also, complementation of BC202s temperature sensitivity and cell division defects are complemented by only four of the eight E. coli DedA proteins (C-group; yqjA, yghB, yohD, and yabI), the remaining four were subsequently classified as non-complementing (NC-group; ydjX, ydjZ, yqaA, and dedA). The identification of the C- and NC-groups within E. colis DedA proteins, lead to the generation of another mutant series in which the NC-group and the C-group proteins were independently deleted, to aide in the continued understanding of this essential and redundant membrane protein family.

The work presented in this dissertation focuses on the temporal regulation of telomerase complex assembly in the yeast Saccharomyces cerevisiae as mediated by protein degradation of a core subunit during G1 phase of the cell cycle. I have discovered that the Est1 protein undergoes rapid degradation only during G1 phase of the cell cycle and that degradation requires function of the Anaphase Promoting Complex (APC), an E3 ubiquitin ligase. Furthermore, release of cells through G1 phase shows that Est1 protein degradation depends upon the APC activator protein Cdh1. Mutational analysis revealed specific amino acids of the Est1 protein that are necessary for degradation. Despite strong evidence for a role of the APC in Est1p stability in vivo, recombinant Est1 protein is not degraded or ubiquitinated in vitro using several different assays. These studies suggest that stability of the Est1 protein in vivo may be indirectly influenced by APC function.

Symbiosis is a process by which two or more distinct organisms interact, whereas speciation is the diversifying process by which one species splits into two. Symbiosis and speciation are not commonly discussed together and can seem to be odd partners in their capacity to operate synergistically in nature. However, the complex community of microorganisms that live in symbiosis within an animal speciesknown as the microbiomehas the capacity to confer new traits and selective pressures that drive speciation. To demonstrate the microbiomes influence in speciation, we present the following evidence using closely related insect species of the model Nasonia. First, the gut microbiome forms phylosymbiotic assemblages in which the relationships of the Nasonia-associated microbiomes parallel the evolutionary genetic relationships of Nasonia. Second, these phylosymbiotic assemblages become irregular in hybrids between the species. Third, the altered microbiome in hybrids is correlated with high rates of hybrid mortality. Fourth, when the hybrids are cured of their irregular microbiota, they are rescued from hybrid mortality. Finally, when the cured hybrids were reinoculated with bacteria, they recapitulated high rates of mortality. We conclude that in this animal complex, the gut microbiome is equally important as host genes in promoting hybrid mortality and thus advancing speciation.

The plasma membrane of eukaryotic cells displays a distinctive asymmetry of the component phospholipids. Type-IV P-type ATPases (P4-ATPases) flip specific phospholipids across the membrane bilayer to establish and maintain this membrane asymmetry. P4-ATPases are closely related to ion-transporting P-type ATPases that use a single site within the center of the transmembrane domain to select and transport their substrates. This single binding site is spatially restrictive and will not accommodate ions of the same valence but different ionic radius. Fitting a phospholipid into this constrictive single binding site to is difficult to imagine and thus, P4-ATPase phospholipid recognition is referred to as the giant substrate problem. This work describes the characterization of the mechanism used by P4-ATPases to select and transport their phospholipid substrates. Surprisingly, P4-ATPases solve the giant substrate problem by using two gates at the protein-lipid interface to select and transport phospholipid, one at each face of the lipid bilayer. A second defining feature of eukaryotes is membrane-bound organelles that serve a multitude of functions. Defined compartments present a challenge for coordinating separate steps in biochemical pathways carried out in distinct organelles. In addition to the role in membrane asymmetry, P4-ATPases play critical roles in communication between the organelles through vesicular-mediated protein transport. Using P4-ATPase variants with altered phospholipid specificity, we identified a role for phosphatidylserine flip in specific transport pathways. Here, we report phosphatidylserine flip facilitates traffic between the trans-Golgi network and the early endosomes, but not in other trafficking pathways within the Golgi-endosomal system. This dissertation has propelled the P-type ATPase and protein trafficking fields forward through a description of a novel P-type ATPase transport mechanism and a physiological role for the phospholipid flip in specific trafficking pathways.

Work presented in this dissertation details structural and biochemical characterization of three yeast alkylpurine DNA glycosylases Mag1 and Mag2 from S. pombe and Mag from S. cerevisiae. I determined high resolution crystal structures all three enzymes and used the structures to initiate a deeper understanding of the sources of substrate specificity and catalytic competence in DNA glycosylases. My study of Mag1 and Mag provided novel evidence that protein-DNA interactions away from the active site can modulate the specificity of DNA glycosylases and a single substitution in the minor-groove interrogating loop is sufficient to alter the preference of one alkylpurine glycosylase for εA to that of a homologous enzyme. The crystal structure of Mag2 along with biochemical and phylogenetic analysis of the Mag genes in related fungal species studies showed that spMag2 cannot form a catalytically competent complex with DNA due to differences in the minor-groove interrogating loop and overall electrostatic surface potential compared to other alkylpurine glycosylases and may have evolved to perform a separate function.

DNA protection during starvation (Dps) proteins are important for bacterial oxidative stress responses. This study aims to understand the roles and characteristics of the two Dps homologs in Deinococcus radiodurans: Dps-1 and Dps-2. Dps-2 contains a predicted signal peptide and in vivo localization of Dps-2 reveals that its location is non-cytoplasmic. β-galactosidase assays show that the Dps-2 promoter is upregulated in the presence of H2O2 and the results from the DNA protection assay show that Dps-2 is able to protect DNA efficiently against reactive oxygen species (ROS). Dps-2 has a C-terminal extension that is needed for assembly into a dodecamer but not for DNA binding. Dps-1 is assembled from six dimers and stoichiometric experiments reveal that the stoichiometry of Dps-1 binding to 22 bp DNA substrate is 1:6, meaning that Dps-1has six DNA binding sites. However, for the 26 bp DNA substrate, the stoichiometry is 1:4, suggesting that the protein can interact with both faces of a DNA duplex provided there are two consecutive major grooves on each face. Furthermore, mutation of the surface arginine (Arg132) causes a decrease in DNA binding, which indicates that this residue is involved in the path of DNA binding of Dps-1 after the initial contact is made with the N-terminus. A model for the mode of Dps-1 binding to genomic DNA is proposed based on these observations. Dps-1 has a unique metal site at the end of the N-terminal extension and mutations of this site cause the protein to exist as a hexamer and this lead to a significant reduction in DNA binding. The mutant protein (Dps-HE) breaks down into dimers and loses its ability to bind DNA upon removal of divalent metals, wherease removal of metals from full-length Dps-1 has no effect on oligomeric state. These findings suggest that the N-terminal metal site is needed for proper assembly, but once the protein oligomerizes to a dodecamer, metals are no longer required to maintain the dodecameric state. These results suggest that the role of Dps-1 might be to organize genomic DNA while the role of Dps-2 might be to provide protection against incoming ROS.

Oyster reefs provide estuaries with ecosystem services including carbon sequestration, shoreline stabilization, refugia for invertebrate biodiversity, and fisheries enhancement, but have been disappearing over the past century due to increasing overharvesting, disease, and eutrophication. The northern Gulf of Mexico has over 400 artificial oyster reefs constructed of various materials created through efforts to restore ecosystem services lost with declining oyster populations. The purpose of this study was to determine how two common artificial reef construction materials (rock vs. oyster shell) at various ages affect ecosystem services, specifically blue crab abundance, the commensal community, and oyster recruitment, as well as to determine the refuge value of oyster reefs. To determine whether the provision of ecosystem services varied over time, rock (n = 7) and oyster shell (n = 6) reefs of varying ages (new < 5 years since construction; old > 5 years since construction) were compared to natural oyster reefs (n = 7) and were sampled twice in the summer of 2011. Sampling devices included baited crab traps, spat settlement plates and commensal collectors. Results indicate no difference between reef category (reef by age) and blue crab abundances, most likely due to their mobility. Diversity, abundance and richness of the commensal community are lowest on old shell reefs. Old rock reefs were most similar to natural reefs in regards to commensal community assemblages, even surpassing them in commensal abundance seasonally. Spat densities and size varied across reef categories, and were more closely correlated with salinity than reef category. To determine refuge value of oyster structure, a mesocosm with a depth gradient and different oyster reef complexities in the deepest end was used. When high complexity shell areas were provided, juvenile crabs were more likely to use these deep water reef refugia when a predator was introduced to the tank; when no shell areas were available, juvenile crabs selected shallow water refuge. The refuge value of oyster reefs, based both on the field survey and laboratory experiment, seems to be the driver of higher commensal abundances, richness, and diversity on older rock reefs, which last longer than old shell reefs which seem to disperse over time in sediments.

The gill, with a large surface area in intimate contact with the environment, is the primary organ of ion and acid-base regulation in fish. These same characteristics make the gill epithelium particularly susceptible to a number of environmental perturbations, including osmotic challenges and metabolic acidosis. For most freshwater fish, the active uptake of the strong ions, sodium and chloride, are intimately linked with the excretion of acid and base equivalents, respectively. However, fish from the genus Fundulus, are unique in their apparent lack of active chloride uptake at the gills. This unique feature makes limiting Cl- loss through paracellular pathways the only practical strategy for these species in tolerating acute exposure to hypoosmotic conditions especially when dietary chloride is limited. We find that Fundulus grandis can dynamically regulate their paracellular pathway in the gill epithelium at salinities approaching fresh water. We observe the significant up-regulation in the mRNA levels of several claudins in the gill and that some of these claudinsexhibit expressional discrepancies between mitochondrion-rich and pavement cells in fish gills, which shows correlations with changes in gill morphology and ion flux rates in fish. Collectively, our data suggest that claudins may play important roles in regulating ion flux across paracellular pathways of Fundulus during osmotic challenges. The linkage between osmoregulation and acid-base tolerance has long been studied in teleost fish. Failure to identify an active Cl- uptake system in freshwater Fundulusindicates the uniqueness of these species in osmoregulation and acid-base tolerance. We find that Fundulus heteroclitus increases Na+ uptake within the first few hours of metabolic acidosis,We also find that metabolic acidosis induces the changes of mRNA levels of several gill claudin proteins, which may play roles in regulating the permeability of the paracellular pathway to strong ions. Our data show Fundulus heteroclitus is capable to cope with great metabolic acidosis within their bodies, which may contribute to their adaptation to internal and external perturbations.

Chlamydomonas reinhardtii (referred to as C. reinhardtii hereinafter) possesses a CO2 concentrating mechanism (CCM) that allows the alga to grow at low CO2 concentrations (100 ppm CO2 in air). This dissertation represents the results from the characterization of three CCM mutants, as well as generation of eleven more potential mutants deficient in the CCM. One common feature seen in photosynthetic organisms possessing a CCM is the tight packaging of Rubisco within the cell. In many eukaryotic algae, Rubisco is localized to the pyrenoid, an electron dense structure within the chloroplast. The first mutant characterized has a highly disorganized pyrenoid. The results indicated that the gene disrupted in this mutant, CIA6, is required for the formation of the pyrenoid. Furthermore this showed that the loss of the pyrenoid correlated with the loss of the CCM. These results supported the hypothesis that the pyrenoid is required for a functional CCM. A second mutant investigated was a revertant of a mutant in the gene PGP1. Initially, the pgp1 strain could not grow under low CO2. But over time, it regained the ability to grow under low CO2 conditions. Data is presented that shows the change in growth phenotype is a result of a second site reversion. The results from this study suggested that another phosphoglycolate phosphatase (PGP2) might play a role in the phenotype reversion. Thirdly, a cell wall deficient strain CC-503 was found to be missing a periplasmic carbonic anhydrase (CAH1). The possible reason for the loss of CAH1, and the resulting consequences of losing CAH1 were investigated in this strain. Lastly, a PCR-based reverse genetics mutagenesis screen was performed to identify more genes involved in the CCM in C. reinhardtii. Eleven potential mutants were isolated, and it was shown that this method could be used on a larger scale in the future to generate mutants missing key CCM genes.