Acinetobacter baylyi ADP1is a naturally competent, non-pathogenic soil bacterium used for the study of natural transformation. Natural transformation is the ability to acquire extracellular DNA and use that DNA as new genetic material. Here, we tested the impact of monovalent cations on transformation efficiency by comparing transformation using LB agar, which contains Na+ ions, to transformation using LBK agar, which contains instead K+ ions. We found no difference in transformation efficiencies using these two types of solid media during transformation. Next, we intended to test the effects of divalent cations on transformation efficiency. But, rates of transformation were so high on both LB and LBK that we first needed to find conditions under which transformation efficiency was <0.1% in order to be able to detect whether the addition of cations would have a positive effect on transformation efficiency. Thus, we reduced the amount of DNA used for transformation, but doing so did not reduce the transformation efficiency. Then, we altered the conditions under which we measured transformation efficiency by adding DNase, which degrades extracellular DNA, in order to restrict the time of DNA availability. However, these manipulations did not reduce the transformation efficiency either. Control experiments verified that the wild type, non-transformed cells were sensitive to the antibiotic chloramphenicol. This control is important because the donor DNA confers resistance to chloramphenicol. Unfortunately, the desired conditions of experimental room were never obtained, and thus a effective comparison of transformation rates due to divalent cation presence was conducted. We conclude that ADP1 cells are extremely competent under all conditions tested.
Acinetobacter baylyi strain ADP1 is a gram-negative bacterium normally studied because of its high competence for genetic transformation and its ability to catabolize plant-derived aromatic compounds. A previous study has identified that the gene cluster ACIAD1969-ACIAD1952 contains genes that may be responsible for potassium tellurite resistance, as well as other proteins that are “hypothetical.” Our goal was to use bioinformatics to investigate this gene cluster and to determine whether it played a role in potassium tellurite resistance as well as twitching motility. Our results indicate that the gene cluster is actually composed of four different operons that play a role in tellurite resistance. We also found that the gene cluster was most likely inherited from horizontal gene transfer, as it is not found in any other Acinetobacter strains. Furthermore, all genes except ACIAD1956, ACIAD1962 and ACIAD1964 are responsible for potassium tellurite resistance in ADP1 and all mutants exhibit twitching motility defects. Our results indicate that the genes in the gene cluster ACIAD1969-ACIAD1952 encode proteins and should no longer be considered “hypothetical.”
Tropical conservation and research has focused primarily on protected areas and has largely neglected the conservation value of the vast and growing agricultural areas. Understanding how species and groups of species persist in deforested areas and react to conversion of tropical rainforest to agricultural land is critical to maximize conservation. I compared bird community composition in four habitats in a mixed agriculture- tropical rainforest ecosystem in Northeastern Costa Rica: organic shade-grown cacao plantation, live fences in silvopasture, riparian forests in a silvopasture matrix, and preserved late successional forest. Point counts over two-months (March-April, 2013) found 167 species from 35 families. Detrended correspondence analysis showed that all three agroforest habitats and the rainforest each sustain a different assemblage of birds, indicating that the varied agricultural landscape in this area supports a higher species richness of birds than any of the individual systems would on their own. Mean number of species identified per point was greatest in live fences, intermediate in riparian forest and cacao , and lowest in the rainforest. New world warblers and the nectar, small insects/spiders diet guild were more abundant in cacao; flycatchers, sparrows, and the ground foraging strata were more abundant in live fences; ovenbirds and omnivores were more abundant in riparian forest; and wrens and the fruits or fruits and seeds diet guild were more abundant rainforest. Many bird species in the agricultural lands occurred only in the shade trees of the cacao plantation, only in the live fences within the pastures, and only in the riparian forest buffers. Given this, legislation and management should continue to require and encourage preserving the forested aspects of the agricultural landscape. To provide maximum large landscape conservation, a varied agricultural landscape must be maintained outside of preserves to promote maximum avian diversity and take advantage of the considerable conservation benefits of many agricultural systems.
Optimal clutch size has been an important focus within evolutionary biology since David Lack’s innovative work in 1947. Prey abundance, typically thought to limit clutch size, may be especially limiting in raptors, since the females contribute minimally to prey provisioning. Studying species with significant energetic constraints may illuminate the relationship between energetics, parental division of labor and clutch size. Flammulated Owls (Psiloscops flammeolus) are a small raptor with prey that is small relative to their body size, further constraining flammulated owls energetically when compared to raptors with larger prey. I hypothesized that female flammulated owls with clutches of three will contribute more to prey deliveries than females with clutches of two, while male prey delivery rates will not vary with brood size. Prey delivery data from 115 flammulated owl nests in the Front Range of Colorado were recorded from 2004-2013. During the second half of the nestling period broods of three received more prey deliveries than broods of two (p < 0.05). Additionally, during the second half of the nestling period no significant difference was found between male and female prey delivery rates for broods of three (p > 0.05). However, among adults with broods of two, males provided significantly more prey than females (p < 0.05). Male prey delivery rate between brood sizes was not significantly different (p > 0.05). These results indicate that broods of three may require greater energy expenditure than broods of two from the female, but not the male parent. While these results pertain to a bird with a small inflexible clutch size, similar research on birds with larger more flexible clutch sizes may reveal how and if clutch size and parental division of labor have co-evolved within avian taxa.
Human induced climate change is expected to increase temperatures in inter-continental regions of Northern America from 2˚ to 4.5˚C. This rapid change alters resource availability and places new stressors on vegetation. In this study I compare plastic avoidance and tolerance responses for two contrasting long-lived forb species (H. quinquenervis and E. speciosus). My experimental site was located in a Colorado subalpine meadow where overhead heaters have warmed vegetation and soil for the past 20 years to mimic expected climate changes. In warmed plots, H. quinquenervis has increased in abundance, size and flowering rate while E. speciosus has decreased in these parameters. Therefore, I expected to observe more tolerance responses in H. quinquenervis than in E. speciosus. To test this hypothesis I assessed photosynthetic CO2 assimilation rates, biochemical photosynthetic limitations and water use efficiency (WUE). Relative to control plots, CO2 assimilation rates were lower in heated plots for both species. However, there were no differences in the photosynthetic limitations derived from these A/Ci curves for treatment, species or their interaction. Lack of significance in these parameters is possibly related to the small sample size. WUE, or carbon gained per unit of water lost, had significantly greater values for E. speciosus than for H. quinquenervis. The ability of H. quinquenervis to maintain low WUE rates under potential water stress may be associated with its root structure as well as its ability to respond through tolerance mechanisms. This study reveals the importance of different stress responses as they relate to species success in a changing climate. By understanding the mechanisms behind differential species success, we can better predict and mitigate future climate changes.
Superpositive green fluorescent protein (GFP) is a designed recombinant bimolecular form of sg100 GFP, the most commonly used variation of native GFP, with a high theoretical net charge, which reduces aggregation, achieved by replacement of aspartic acid residues with arginine and lysine residues. Here, we expressed superpositive GFP as separate N-terminal and C-terminal domains (split superpositive GFP) in order to develop an assay for the detection of proteins binding to one another and reconstituting functional GFP fluorescence.This recovery of fluorescence upon protein pair binding provides an easily observed and semi-quantitatively measurable analogue for the successful interaction of the proteins. The objective of this project was to use the split-superpositive GFP protein complementation assay in conjunction with free or orthogonally reporting inhibitor proteins. We hoped to find that the presence of high affinity free inhibitor proteins would correlate with a decrease in recovered fluorescence when compared to cells in which a lower affinity inhibitor, or no inhibitor, is present. As a secondary objective, we also tested whether an inhibitor conjugated to the N terminal fragment of split cerulean fluorescent protein would be capable of providing an orthogonal cerulean signal upon competitive binding with the C terminal fragment of split-superpositive GFP. Evaluating time dependent evolution of fluorescence within cells expressing this system using flow cytometry could provide a novel method of screening macromolecular protein-protein interactions and inhibitors of these interactions rapidly and easily.
Dietary folate deficiency is associated with the functional decline of cells, tissues, and organs and the development of acute lymphoblastic leukemias in children. In mice models, leukemogenesis is initiated by Bcr-Abl oncogenic translocations promoted as a result of folate deficiency. Conventionally, cancer is thought to be the result of accumulations of oncogenic mutations over time. Through the application of the Adaptive Oncogenesis model we hypothesize that folate deficiency is likely to reduce the fitness of stem and progenitor cell populations. This reduction in fitness may lead to increase selection for oncogenic mutations that can partially alleviate folate deficiency fitness defects, thereby promoting the initiation of cancers. I tested this hypothesis by mimicking folate deficiency in B-progenitors by using methotrexate (MTX) to target DHFR (dihydrofolate reductase) an enzyme that converts dihydrofolate (i.e. DHF, a folic acid derivative) into tetrahydrofolate (THF) in the folate pathway. I found that in vitro, Bcr-Abl expressing B-progenitors offer partial protection in response to MTX. Furthermore, within a competitive environment, the decline in the fitness of folate deficient B-progenitors promote selection for Bcr-Abl expressing B-progenitors as a result of folate-dependent alterations of the fitness landscape. Expression of the Bcr-Abl oncogene provides a greater competitive advantage compared to control BaF3 Parentals and BaF3 vectors, but only in the presence of methotrexate. These studies establish the development of a cost effective, high-throughput in vitro system to assay for various required components that control folate metabolism and nucleotide synthesis in B-progenitors. Future research will focus on figuring out the mechanisms by which Bcr-Abl B-progenitors are advantageous in a folate deficient environment.
Acinetobacter baylyi ADP1 is a gram-negative soil bacterium that exhibits competence and twitching motility. DNA uptake is achieved via the Type IV Pilus competence machine and twitching is performed by Type IV pili. Homologues of Type IV pili proteins are involved in transformation in a variety of bacteria. The similarities between proteins involved in DNA uptake, Type IV pilus systems and type II protein secretion systems suggests that they belong to evolutionary related systems containing cell envelope spanning structures with conserved architecture and core components. As many competence proteins of ADP1 are related to structural subunits and biogensis proteins of Type IV pili, a key question is whether Type IV pili of ADP1 are directly involved in DNA uptake and binding. Or, do the pilin-like components of the transformation system make up a completely different structure? Many bacteria can perform natural transformation; however, our knowledge regarding the structures and mechanisms needed for DNA uptake is scarce. Thus, our research involved determining which genes are needed for competence, which are used for twitching motility and which are possibly involved in both functions in ADP1. In order to test each protein’s role, tdk-kan knock out mutants were created and the mutants were compared to the wild type. An existing library of proteins predicted to encode various parts of the Type IV pilus with knock out genes was used. Our results showed that the majority of tested genes are needed for both competence and twitching, suggesting a physiological relationship. Specifically, mutants with a greater twitching ability were also more competent.
Aedes aegypti is the primary disease vector for both Dengue and Yellow Fever. Climate change is affecting the natural environment of these mosquitoes, yet the extent to which individuals and populations can physiologically respond to environmental stressors is unknown. Understanding the mechanics of A. aegypti stress responses has important implications for predicting both mosquito and disease distribution patterns in the context of a rapidly changing global environment. In this research, the genetic response of adult female A. aegypti mosquitoes to dehydration stress is investigated using quantitative PCR. Homologs of four physiologically relevant genes (Frost, Desat2, HSP70 and Pepck) are examined in A. aegypti. Mosquitoes were subjected to acute desiccation stress and then analyzed via qPCR to determine the extent to which these genes' expression patterns were altered. Altered gene expression in response to desiccation stress was observed for HSP 70, while evidence suggesting the evolutionary divergence of Frost was also uncovered. These results provide clues as to which physiological mechanisms are utilized by A. aegypti to mediate survival in desiccating environments. Genetic indicators of these mechanisms can be used in comparative studies against geographically distinct populations to generate an understanding of stress resistance mechanisms in A. aegypti as a function of geography and environment.
The mechanism of vesicular trafficking is a process that is not well understood, but it is critical to the operation and function of a cell. Rab proteins guide anterograde (ER to plasma membrane) and retrograde (plasma membrane to ER) directional vesicular movement, but most Rab proteins have been shown to have varying trafficking roles with membrane-bound vesicle movement, tethering, and fusion specific to the cargo within the vesicle. This study aimed to identify Rab proteins that are involved in anterograde trafficking of human gonadotropin-releasing hormone receptor (GnRHR). Plasmids encoding Rab GTPase DNA, along with human GnRHR DNA, were transfected into Cos-7 African green monkey kidney tumor cells in vitro. Cells were grown in tritium inositol and upon stimulation of GnRHR, inositol phosphates (IP) containing radioactive markers were synthesized. A radioimmunoassay was conducted to determine total presence of inositol phosphate (IP), a marker that correlates with GnRHR presence at the plasma membrane. Rab6 and Rab3a were identified as significantly increasing IP levels, an indication of higher presence of GnRHR at the cell membrane and anterograde activity. Moving forward, further research will be pursued to find additional Rab GTPase proteins involved in anterograde trafficking and their specific function and localization. Understanding the pathway of GnRHR to the plasma membrane could improve therapeutic treatments for hypogonadotropic hypogonadism, a disease caused by loss-of-function of GnRHR due to a failure of expression at the cell membrane.
Acinetobacter baylyi is a gram-negative soil dwelling bacterium. The strain ADP1 is highly competent which allows for easy manipulation of its genes. The genes of interest here are a set of the genes encoding a type VI secretion system (T6SS), namely tssG, tssF, tssE, tssB, and tssC. This T6SS is homologous to the bacteriophage tail. The tail of the bacteriophage is used to inject DNA into a bacterial cell. Therefore, we hypothesized that the T6SS could be used to release DNA into the environment. Over the course of this research, we investigated ADP1 bacterial cells that contained knockouts of the various parts of the T6SS. The knockouts were used to determine which, if any, of the parts of the T6SS play a role in DNA release. We also tested the mutants for differences in growth rate and survival in long-term stationary phase (LTSP). LTSP is a phase in which 99% of the cells die off and the remaining 1% begin to eat waste and dead cells to survive. We examined survival in LTSP because another T6SS gene, vgrG (ACIAD0167), is known to be expressed during LTSP (Lostroh and Voyles, 2010). The gene is also necessary for survival during LTSP (Stanley and Lostroh, 2010) and twitching motility (Nguyen and Lostroh, 2013). We discovered that the core T6SS genes are not needed for a normal growth rate during exponential phase or for DNA release. However, we were unable to definitively determine if tssG, tssF, tssE, tssB, and tssC are necessary for survival during LTSP due to poor survival rates of the wild-type and mutants, likely caused by evaporation of water over the course of the experiments. Further research will be performed to determine which secretion system if any is responsible for DNA release and if tssG, tssF, tssE, tssB, and tssC are necessary for survival during LTSP.
Lipoprotein lipase plays a central role in the metabolism of lipids in all vertebrate organisms. It catalyzes the hydrolysis of triglycerides from circulating chylomicrons and very low density lipoproteins (VLDL) into free fatty acids and glycerol. LPL also serves as a bridging ligand between the endothelium and lipoproteins facilitating the uptake of neutral lipids. Deficiency in LPL causes hyperlipidemia which when untreated can result in serious disorders including pancreatitis. LPL is most abundant in muscle and adipose tissue, however, it is also present in neuronal tissue where effects on metabolism and organism behavior have been documented. Several mutations in LPL have been studied and are often associated with increased risk for atherosclerosis and hyperlipidemia, although a gain of function mutation has also been observed. To help understand the effects of these mutations on enzyme function and neuronal cell metabolism several recombinant LPL mutants were created. Included was a catalytically inactive mutant (Gly188Glu), a mutation effecting lipid substrate binding Trp393Ala/Trp394Ala, a mutation associated with atherosclerosis in a GWA study (Asp9Asn), and a gain of function mutation (Ser447Ter). These mutants were transduced into a mouse neuronal cell line using a retroviral vector. LPL activity and neutral lipid accumulation were then studied in the transduced neurons. This was conducted utilizing an LPL assay using a tritiated triolein emulsion as substrate. In addition, catalytic independent lipid uptake was examined through the use of the Gly188Glu mutant. Intracellular TG content and lipid uptake were increased with the overexpression of the mutant suggesting a role for LPL in lipid uptake independent of TG hydrolysis. Additionally, mLPL C-His was purified using a nickel column in order to help create a new antibody against it.
The ETS family of transcription factors is made up of 30 evolutionarily-related proteins that are characterized by the DNA-binding ETS domain, which is highly conserved throughout the family. All ETS proteins bind to a consensus 5’-GGA(A/T)-3’ DNA sequence motif, yet regulate transcription at a diverse array of genomic targets, so the mechanisms of their regulation are of particular interest. The members of the ETV1/4/5 subfamily are aberrantly expressed in multiple cancers, and display a biochemical phenomenon called autoinhibition in which intramolecular interactions decrease the affinity with which the protein binds to DNA. This project quantitatively maps the autoinhibition of ETV5, and establishes relationships between autoinhibitory structural elements and corresponding effects on DNA affinity. I show that ETV5 contains autoinhibitory sequences both N- and C-terminal to the ETS domain. Additionally, I demonstrate that both helices and unstructured regions C-terminal to the ETS domain play a role in autoinhibition. Finally, I establish that a single residue, Met457, plays a role in mediating autoinhibition in three separate C-terminal truncations.
There are hundreds of phylogenetically diverse soil bacteria with the capacity to grow on a wide range of antibiotics as their sole carbon source. Some of these bacteria are closely related to human pathogens. The present study evaluates whether there is a barrier that might prevent the penicillin catabolism genes from transferring from the penicillin catabolism strain ABC 07 to the Acinetobacter baylyi strain ADP1. Because of its natural competence and its close relation to the human pathogen Acinetobacter baumanni, ADP1 is an ideal model for the current investigation. ADP1 was transformed with the genomic DNA of ABC 07, Sau3A1 genomic libraries, and Sau3A1 genomic plasmid libraries to test this barrier. None of these methods transformed ADP1 to be a strain with a penicillin catabolism phenotype. In this regard, recent research (Walsh et al., 2013) challenges the concept of bacteria subsisting on antibiotics and argues that the SCS selective medium used in the original study (Dantas et al, 2008) contains 15 mg/L EDTA, which could be the carbon source that sustained the growth of these “antibiotic-eaters.” The present study also tested and verified that ABC 07 can subsist on penicillin, but not EDTA. These results suggest that antibiotic catabolism genes cannot be readily transferred from antibiotic catabolism strains to other bacteria. Future research related to antibiotic catabolism phenotype should attempt computational approaches and system-level molecular methods to identify antibiotic catabolism genes and metabolic pathways before further characterizing their clinical and ecological implications.
Urbanization and anthropogenic development across North America are contributing to habitat loss and fragmentation. Urbanization also alters surface water systems, resulting in the elimination, alteration, and creation of aquatic ecosystems. Habitat loss is one factor contributing to the current native bee and honeybee (Apis mellifera) population declines across the continent. Previous studies on the effect of urbanization on bee populations have produced conflicting results, which suggest that further research is required. The effect of surface water availability on bee populations is not well studied. Using bee bowl traps and sweep net sampling techniques in household yards across the Twin Cities in Minnesota, I assessed bee abundance and bee community composition across an urban to rural gradient using housing density as a measure of degree of urbanization. I also examined and compared bee communities in yard sites both near to and far from major surface waters. Specifically, I tested the hypothesis that bee community assemblages are affected by both housing density and proximity to water, independently. I found no significant difference in bee abundance across the urban to rural gradient or at varying distances from water. However, I found a positive correlation between yard size and bee abundance and a significantly different community composition of bees near to and far from water. The results of this study imply that bee populations are not affected by housing density alone, and that other factors, such as habitat patch size as measured by yard size, may be contributing to reported declines in bee populations. Results also imply that altering surface waters in urban areas can impact bee community composition. These results can help guide future studies and inform urban planning and surface water alteration methods in order to conserve bee populations.
Bats are the most species richness mammalian order in the tropics. Throughout the world, bats comprise 25% of mammalian species and this percentage increases to over half along the equator. The large ecological impact of bats, through insect control and pollination for example, is related to their species diversity and abundance in the tropics. Because of this large impact, it is important to study the effects of habitat fragmentation gradient including agriculture on these bat populations. I examined the differences in insectivorous bat species diversity and activity in the habitat gradient between the tropical rainforest in Tirimbina Biological Reserve in La Virgen de Sarapiquí, Heredia, Costa Rica and the nearby organic pineapple farm, Finca Corsicana. I hypothesized a decrease in species richness and activity with an increase in human disturbance from the forest, to the edge of the forest, to the pineapple farm (activity is defined as the number of passes per minute). I also predicted that the different bat families would show a difference in habitat preferences due to the habitat specialist versus generalist classification system, which includes their distinct flight and eating patterns. Finally, I expected a difference in species diversity and activity throughout the night and a variation between collection days. Over four weeks in March and April 2013, I placed ultrasonic recorders at 12 different sites to pick up the bats’ calls. I then analyzed the recordings to identify the species. I found four families and 22 different species of bats. The results showed a significant decrease in the number of bat passes (the number of times a bat passes the recorder per minute) in the pineapple farm, in comparison to the edge, and the forest. Along with this main conclusion of all bat species, the Emballonuridae foraged mostly in the edge habitats, the Molossidae were spread out between the edge and forest, and the Vespertilionidae were found evenly among all three habitats. There was not a significant difference in bat activity through out the night, however there was a significant difference in the mean number of passes per week and per day. Overall, my results suggest that maintaining forest and edge habitats are important to maintaining species richness of bats in tropical rainforests.