Upon exposure to anoxia, encysted embryos of the brine shrimp, <i>Artemia franciscana</i>, enter a severely depressed metabolic state, the transition into which is facilitated in part by one of the largest intracellular acidifications ever reported for a eukaryotic organism. However, the endogenous origin of this pH shift remains largely unexplained. We hypothesize that the unexplained acidification is produced by a net flux of protons into the cytoplasm from compartments acidified by a V-type proton pump (V-ATPase) during aerobic development. Northern blots demonstrate expression of the V-ATPase subunit-B mRNA during early development, while Western blots demonstrate the expression of at least 6 constituent subunits of the V-ATPase in both heavy membranes and microsomal vesicles. Inhibition of embryo hatching with the highly specific V-ATPase inhibitor, bafilomycin A1, confirmed a requirement for V-ATPase activity during the anoxia tolerant stages of development. Given that the V-ATPase is responsible for acidification of intracellular compartments in eukaryotes, these data indicate the presence of compartmentalized proton stores in <i>A. franciscana</i> embryos. ³¹P-NMR studies using intact embryos demonstrate that V-ATPase inhibition with bafilomycin A1, severely limits intracellular alkalinization during recovery from anoxia without affecting the restoration of cellular nucleotide triphosphates. Based on these data, it appears that oxidative phosphorylation and ATP resynthesis can only account for the first 0.3 pH unit alkalinization observed during aerobic recovery from 1 h of anoxia. The additional 0.7 pH unit increase requires proton pumping by the V-ATPase. Furthermore, aerobic incubation with the protonophore, carbonyl cyanide 3-chlorophenylhydrazone, produces an intracellular acidification similar to that observed after 1 h of anoxia, and subsequent anoxic exposure yields little additional acidification. When combined with protons generated from net ATP hydrolysis, these data show that the dissipation of proton chemical gradients is sufficient to account for the reversible acidification associated with quiescence in these embryos. Whole embryo respirometry demonstrates that V-ATPase activity and processes immediately dependent on that activity constitute approximately 31% of the aerobic energy budget of the preemergent embryo. Downregulation of such a costly transporter would be essential in order to attain the level of metabolic depression observed in the whole embryo under anoxia.

Newly forming species that have differentiated in allopatry may evolve numerous barriers that prevent the interbreeding when they come back into contact with each other. The objective of this dissertation is to evaluate some mechanisms of prezygotic reproductive isolation in the D. pseudoobscura subgroup. I begin by evaluating how the evolution of female preferences and male sexual characters lead to reinforcement between Drosophila pseudoobscura and its congener D. persimils. In particular, I will evaluate two alternative hypotheses; Preference Evolution and Discrimination Enhancement, to determine how selection reduces hybridization between these sister species. Both hypotheses predict a reduction in the overlap of male traits and female preferences in hybridizing populations; however, the target of selection differs between the two. Next, I will discuss reproductive isolation as a result of competiton between gametes, in particular conspecific sperm precedence. Until this study, patterns of sperm precedence had rarely been examined between divergent populations or subspecies within a species. I will evaluate conspecific sperm precedence and its role in reproductive isolation between two subspecies: Drosophila pseudoobscura pseudoobscura and D. p. bogotana. The final portion of this dissertation examines the rapid evolution of some proteins potentially tied to the evolution of reproductive isolation. I focus on some seminal fluid proteins that may play a role in the reproductive isolation of Drsosphila species. In particular, I examine the rapid evolution of accessory gland proteins in the D. pseudoobscura subgroup by looking for the signature of positive selection in the genes that encode them. I will also evaluate the roles of insertion / deletion mutations in the evolution of these proteins. Together, the chapters of this dissertation contribute to the understanding of three forms of prezygotic reproductive isolation and their roles in speciation.

The complex life style of streptomycetes requires that the appropriate program of genes be expressed at the appropriate times. For example, conjugation occurs solely within the substrate mycelium stage of development while antibiotic production occurs in the aerial mycelium. Thus, complex regulatory mechanisms involving numerous modes of control have evolved that confer temporal regulation of functions governing these expression programs. Conjugation of the Streptomyces lividans plasmid pIJ101 utilizes only seven plasmid functions in its transmission. Two of these, tra and clt, are essential while three additional functions, spdA, spdB and kilB augment the process, with KorA and KorB regulating the transfer and spread functions. The transmission operon of pIJ101 is unique, in that multiple layers of transcriptional and posttranscriptional control converging to implement tight control of the transfer and spread functions, specifically the potentially lethal kilB gene product. When expressed unregulated, as a chromosomally integrated gene, kilB transcription decreases during the later stages of development, in sharp contrast to the temporally increasing pattern of KilB protein. However, when expressed on pIJ101, the kilB promoter is largely, if not completely repressed, and expression of kilB requires transcription readthrough from upstream. Furthermore, readthrough transcription terminates within a 105 base-pair intergenic region prior to kilB. Interestingly, kilB-operator-bound KorB repressor appears to act, at least in part, as an attenuator of operon transcription, perhaps by physically barring transcription elongation. Finally, the formation of a stem-and-loop in the readthrough transcript within the intercistronic region appears to promote antitermination of operon transcription to counteract the effects of the KorB roadblock. Regulation of antibiotic production in Streptomyces typically involves the streptomycete global regulatory mechanism bldA. The sweet potato pathogen Streptomyces ipomoeae strain 91-03 produces a bacteriocin-like antibiotic, ipomicin. In liquid culture, ipomicin is produced in low concentrations throughout exponential phase followed by a dramatic 10-fold increase as the culture enters stationary phase. In contrast, transcription of the ipomicin structural gene ipoA decreases as the culture ages. The contrasting patterns of ipoA transcription and ipomicin production, coupled to the fact that ipoA contains a TTA codon in its leader sequence, makes ipomicin a strong candidate for bldA regulation.

The cloud forest sugar maple, Acer saccharum subsp. skutchii, occurs as five disjunct populations, four in Mexico and one in Guatemala. I assessed the current status, distribution, and environmental relations of forests containing these populations, and I compared the species composition of these forests with other temperate and cloud forests in North and Central America. I gave special emphasis to a recently discovered population in Talpa de Allende, Jalisco, Mexico, by assessing its tree richness and generic composition in a continental context. In the five studied cloud forest sugar maple populations, basal area of all trees ¡Ý1 cm DBH varied between 25.7-52.2 m2ha-1, and density ranged from 990-2929 trees per ha. A. saccharum subsp. skutchii represented 7-43% of the total basal area and 1-16% of the tree stems. Bray & Curtis ordination of cloud forest sugar maple populations indicated that most of the variation in relative tree abundance could be explained by soil characteristics and presence of canopy gaps. More cloud forest sugar maples occurred in sites with higher soil moisture (r=0.716). In contrast, a NMS ordination indicated that the majority of the variance in community composition of all temperate and cloud forests analyzed was related to latitude, elevation, and precipitation. Tree species richness of Talpa de Allende and 14 other temperate and cloud forests around the world was significantly different (F=27.53, p=<0.0001). Species richness of forest in Asia and Talpa de Allende did not differ. In addition, generic composition was similar for forests in Asia and Talpa. Based on NMS ordination and Ward&39;s classification, I hypothesize that six forest sites in Jalisco, Tamaulipas, Veracruz, and Hidalgo (Mexico) contain a unique and ancient flora, were connected and shared species before the Pleistocene, and currently function as tree refuges of that ancient flora. Based on the limited distribution of cloud forest sugar maple and its small number of extant populations I propose the inclusion of A. saccharum subsp. skutchii in the IUCN Red List Catalog and as Endangered in the Guatemalan Species Red List. In addition, I propose the creation of a 56,394.9 ha Biosphere Reserve in Talpa de Allende.

Studies of cophylogeny greatly increase understanding of associations between hosts and their parasites. This study uses molecular data to test the hypothesis that members of two rodent families (Geomyidae and Heteromyidae) and their associated lice (Geomydoecus and Fahrenholzia, respectively) show a statistically significant pattern of cophylogeny. Both host groups are generally solitary organisms and both louse groups are obligate ectoparasites, often exhibiting extreme degrees of host specificity. This intimate and potentially long-term association likely has resulted in coevolutionary adaptations and counter adaptations on the part of both symbiotic partners. Phylogenetic analysis of chewing lice (Geomydoecus) reveals two major clades corresponding to the G. coronadoi and G. mexicanus species complexes. These louse complexes are reciprocally monophyletic, and each clade within each complex parasitizes a different species of pocket gopher. Both louse species complexes exhibit a significant pattern of cophylogeny when compared to their hosts. The mitochondrial COI gene of lice of the G. coronadoi complex is evolving approximately 2 -3 times faster than the COI gene of their hosts, whereas the COI gene of lice of the G. mexicanus complex is evolving at roughly the same rate as the same gene of their hosts. Future analyses are necessary to determine why evolutionary rates in these two parasite lineages differ. The phylogenetic analysis of sucking lice (Fahrenholzia) resolves relationships among 11 of the 12 currently recognized species and identifies several possible cryptic species. Although there is conflict among the basal nodes of the host and parasite phylogenies, cophylogenetic analysis reveals significant topological congruence between these lice and their heteromyid hosts. The mitochondrial COI gene of Fahrenholzia lice is evolving roughly 1.6 times faster than the COI gene of their hosts, but additional comparisons of molecular rates are necessary to determine if this rate difference is shared by other groups of sucking lice and their hosts. Results of this study indicate that a combination of tree-based, distance-based, and data-based methods should be used in cophylogeny analyses. The final chapter of this dissertation presents a compilation of mammal-louse associations reveals and offers a preliminary assessment of sucking louse prevalence and abundance on heteromyid rodents.

Sequential large-scale disturbances may produce interacting effects that differ from those predicted for each disturbance in isolation. These non-additive effects can strongly influence the composition and structure of plant communities. Hurricanes and natural lightning-season fires are large-scale, frequent disturbances in southeastern savanna-forest landscapes. Although interactive effects have been proposed, my research is the first to develop and experimentally test mechanistic hypotheses for hurricane-fire interactions. I develop a predictive conceptual model for interacting disturbances. I propose that hurricane-fire interactions depend on the relative timing of disturbances and the duration of effects. To predict the conditions under which hurricane-fire interactions are expected, my mechanistic hypotheses incorporate rates of fine fuel re-accumulation after a fire relative to decomposition of fine and coarse woody debris after a hurricane. This model suggests that the probability for disturbance interactions varies across savanna-forest landscapes. I predict that 1) hurricane-fire interactions are most likely in savannas, 2) they are least likely in forests, and 3) they may influence ecotones between savannas and forests by changing species composition and structure. Based on predictions, I implemented an experimental study in savanna-forest ecotone to test hypotheses of interactive effects. I hypothesized that effects of lightning-season fires differ when fires occur alone compared to when fires are preceded by hurricanes. I simulated two main effects of hurricanes as treatments canopy disturbance and fine fuel deposition by removing canopy trees and manipulating fuel loads. Compared to unaltered controls, I predicted hurricane treatments would influence fire intensity and vegetation response. Both canopy disturbance and fuel addition influenced the behavior of subsequent fires. In addition, the two main hurricane effects interacted to increase maximum fire temperatures. High fuel loads and fire resulted in disturbance interactions that reduced stem density and species richness of woody plants. Reduced hardwood density in areas of locally intense fires may decrease competition between species and increase establishment of pines and other fire-resistant species. Thus, hurricane-fire interactions influence vegetation structure in savanna-forest ecotones. Furthermore, over longer time scales interactions may result in landscape-level changes in southeastern savanna-forest ecosystems.

The MarR family of transcriptional regulators is an important group of prokaryotic DNA binding proteins. As the family name implies, multiple antibiotic resistance, members of the MarR family often regulate the expression of resistance genes to multiple antibiotics, organic solvents, household disinfectants, detergents, and oxidative stress agents. Most MarR members act as transcriptional repressors and exist as homodimers in both free and DNA-bound states. DNA-binding is mediated via a winged-helix fold and is often relieved by anionic lipophilic ligands. Deinococcus radiodurans R1 was found to encode a 181 residue MarR homolog designated HucR (hypothetical uricase regulator). Biochemical evidence has shown that HucR negatively regulates expression of uricase and this repression is attenuated by the binding of uric acid, which is the natural substrate for uricase. In this study we present the crystal structure of HucR determined to 2.3 Å in the absence of ligand. In addition, a second crystal form of HucR was determined to 2.9 Å in which three dimers were observed in the asymmetric unit. Unlike the crystal structure of the MarR homolog, MexR, HucR does not display large conformational heterogeneity between dimers. Furthermore, superpositioning of the HucR dimer with the crystal structure of the OhrR dimer complexed with DNA suggests that HucR is in a "DNA ready" confirmation in which the lobes of the DNA binding domains are in a position compatible with DNA binding, with the exception of minor localized conformational changes needed at the amino termini of the recognition helices. This is in contrast to what is observed when comparing the crystal structures of the DNA-bound and unbound OhrR, in which there is a significant displacement of the DNA binding domains as a result of conformational changes that originate at the dimerization interface. The crystal structure of HucR in the absence of either ligand or DNA suggests that HucR is likely to be fixed in a "DNA ready" conformation. Thus, the crystal structure of HucR has given new insight into the MarR family of transcriptional regulators, proving that although these family members share similar structural folds, their mechanisms for transcriptional regulation are likely very specialized.

The sale of small turtles is banned from the US market due to concerns about their excretion of Salmonella spp. To produce a safe pet for the export market, the Louisiana pet turtle industry uses 1000 μg/ml gentamicin sulfate baths to eradicate Salmonella spp. from turtle eggs. In 1999, we analyzed bacterial samples recovered from turtle farms and found that strains of Salmonella enterica subsp. arizonae and other bacteria such as Salmonella enterica subsp. enterica, Enterobacter cloacae, Citrobacter freundii, Pseudomonas aeruginosa and Stenotrophomonas maltophilia, were resistant to high concentrations of gentamicin (>2000 μg/ml) and to other aminoglycosides. The goal of this study was to identify the gene(s) contributing to the high-level gentamicin resistance phenotype observed in bacteria from environmental samples with turtle farming activity, particularly the salmonellae, and to estimate their incidence in these bacteria, as well as to explore the molecular elements that contribute to the dissemination of gentamicin resistance in this context. R-plasmids from gentamicin resistant strains were transferred by conjugation and transformation to naïve Escherichia coli cells. Cloning and sequencing of the gentamicin resistance determinants on these plasmids revealed the presence of aminoglycoside acetyltransferase genes aac(3)IIa and aac(3)VIa. Multiplex PCR assays showed that every gentamicin resistant isolate carried either one of these acetyltransferase genes. Pulsed-Field Gel Electrophoresis and restriction enzyme digestion analysis of R-plasmids carrying these genes revealed plasmids with sizes ranging from ca. 45 kb to 145 kb, with different restriction profiles, supporting their presence on mobile molecular elements. In effect, gene aac(3)VIa was present as a gene cassette of a class 1 integron. Furthermore, evidence suggests that gene aac(3)IIa may be encoded as part of a complex class 1 integron. The presence of aac(3)IIa and aac(3)VIa in mobile molecular elements explains the distribution of these genes in the different plasmids and organisms described in this study. Further studies are required to characterize these molecular elements, and to detect genes which may be co-selected in the presence of gentamicin. This work is one of the first to report dissemination of high-level gentamicin resistance in Gram-negative bacteria, particularly in environmental isolates of Salmonella enterica subsp. arizonae.

Arabidopsis thaliana is an organism that can be used as a model for most of the processes that occur in flowering plants. The leaf hairs, or trichomes, of Arabidopsis thaliana are macroscopic single cells that have been used as a model system for cell fate determination, cell expansion, cell cycle regulation, cell wall deposition, as well as other processes. Initiation of the trichome cell fate is controlled by a complex of genes including GLABRA1 (GL1), TRANSPARENT TESTA GLABRA (TTG), and GLABRA3 (GL3). This work examines the role of GL3 in trichome initiation and uses plants expressing varying levels of GL3 to determine if genes involved in trichome development are regulated by GL3. Though several genes are given a cursory examination, the regulation of two genes, an α-carbonic anhydrase and a novel cell-cycle regulator called SIAMESE, are given a thorough examination. The α-carbonic anhydrase At2g28210 was previously not known to be involved in trichome development. Its involvement in trichome development was discovered with the aid of an enhancer trap line with robust reporter gene expression in developing trichomes. Pharmacological studies indicate that this α-carbonic anhydrase may play a role in trichome expansion. SIAMESE (SIM) was first identified in a mutant screen in the Larkin lab. This dissertation demonstrates that this gene encodes a novel type of cell-cycle regulator with several homologs in Arabidopsis and other plant species. SIM and one of its homologs in Arabidopsis were shown to be expressed in a trichome-dependent manner. These investigations shed new light into the molecular process of trichome development.

Vibrio vulnificus is a marine bacterium capable of causing severe and fatal illness in susceptible people. The primary virulence factor is a capsular polysaccharide (CPS) that allows the bacteria to evade the host immune system. Two phenotypic forms were previously known to exist, opaque (virulent) and translucent (avirulent), and had been shown to undergo spontaneous phase variation, each switching to the other phenotype. Provided here is the first report of a third phenotype, rugose. The rugose variant is dry and wrinkled compared to the smooth colony morphology of the opaque and translucent forms. Both opaque and translucent strains produce rugose variants that are similar in some ways, but show important differences between the two. Rugose V. vulnificus produce copious amounts of rugose exopolysaccharide (EPS) that allows them to form well-defined biofilms in attachment to glass, as a pellicle on the surface of broth, and as a 3-dimensional colony of distinct architecture. The rugose forms survived better in normal human serum than did their opaque and translucent parents, while only the translucent parent and its rugose variant survived exposure to chlorine. It is proposed that opaque-derived rugose variants also produce CPS, in addition to EPS, and that it is the CPS that actually confers susceptibility to chlorine. Five genes associated with CPS and/or EPS production were expressed by all phase variants at all points of the life cycle. Also reported here is a group of genes, the rugose gene cluster, differentially expressed by the three phenotypes. Opaque and translucent forms expressed genes in this cluster at low levels or not at all, while the rugose variants expressed all of these genes at higher levels throughout exponential growth. As biofilms have been shown to protect bacteria from adverse environmental conditions and the rugose variants produce robust biofilms, this form may contribute to the survival of this bacteria in its natural environment. Future study of these genes should provide insight into the expression and regulation of the rugose phenotype and the resulting biofilms, as well as survival and virulence of V. vulnificus.