Browsing by Subject "Biology, Microbiology"

Human papillomavirus (HPV) infection of mucosal epithelium by `high-risk' HPV types has a prominent role in the development of anogenital intraepithelial neoplasias and carcinomas. Human epithelial cells transformed with the HPV E6 oncoprotein survive even under conditions that normally lead to cell apoptosis. This phenomenon has been attributed to HPV E6's ability to promote the degradation of the tumor suppressor protein p53. More recently, it has been demonstrated that HPV E6 contributes to activation of the NF-kB pathway. NF-kB is a transcription factor involved in the regulation of genes associated with cellular proliferation, apoptosis and inflammatory responses. In addition to p53 suppression, HPV E6 modulation of NF-kB activation presents another mechanism for HPV-driven tumorigenesis. However, it was not known how HPV E6 promotes NF-kB pathway activation. To address how HPV E6 leads to NF-kB activation, we identified an association between HPV E6 and the human cylindromatosis gene product (CYLD). CYLD is an endogenous inhibitor of canonical NF-kB activation. We showed that HPV E6 proteins could precipitate CYLD in vitro using a co-immunoprecipitation assay. Demonstrating that HPV E6 and CYLD proteins bind each other raised the possibility that this binding relationship would have a functional effect upon the NF-kB pathway by altering CYLD-mediated suppression of NF-kB activation. To identify HPV E6 functional relationship with CYLD and to determine how HPV E6 activates the NF-kB pathway, we transfected cells with either HPV E6 expression vectors containing the high-risk HPV type 16 E6 or the low-risk HPV type 11 E6 along with a CYLD expression vector. We showed HPV16 E6 expression in 293 cells blocked the ability of CYLD to inhibit CD40 ligand-stimulated NF-kB activation. Interestingly, HPV11 E6 was unable to inhibit CYLD mediated suppression of NF-kB in our system. CYLD had previously been shown to suppress NF-kB activation by removing stimulatory lysine 63-linked ubiquitin chains from TRAF2. We found CYLD expression in 293 cells leads to dose-dependent reduction in TRAF2 levels. This CYLD-mediated loss of TRAF2 is inhibited by co-expression of high-, but not low-risk E6 proteins. It was known that CYLD phosphorylation in vivo suppresses CYLD deubiquitination actions of canonical pathway proteins; we therefore tested the extent of CYLD phosphorylation when co-expressed with HPV E6, and discovered that CYLD phosphorylation was increased in the presence of HPV E6. This compilation of experiments suggests that with HPV16 E6 binding to CYLD, the E6 protein blocks CYLD-mediated TRAF2 loss and thereby TRAF2 is available to activate the canonical NF-κB pathway. Blocking HPV E6-mediated NF-kB activation may prove beneficial as a means for designing therapies that inhibit HPV-mediated tumorigenesis. The differences we detected between HPV11 E6 and HPV16 E6 are supported by other studies that showed E6 protein variations account for molecular and clinical differences among HPV infection outcomes. Similarly, there exist intratype E6 variations in HPV16. We obtained cervical specimens from patients with cytopathogenic changes consistent with the onset of cervical dysplasia infected with HPV16 E6 and the human immunodeficiency virus. We hypothesized the immunocompromised individual may harbor unique HPV16 E6 variants. Using PCR detection methods to amplify the HPV16 E6 DNA and sequencing technology, we identified that some of the samples indeed had nucleotide polymorphisms, resulting in amino acid sequence changes. However, the HPV E6 variants we detected were previously described, and fit know geographic HPV clades. Some of the HPV E6 variants we observed are suggested to be associated with progression to cervical cancer, but further evaluation is required.

The lipid A component of lipopolysaccharide (LPS) in the nitrogen-fixing plant endosymbionts Rhizobium leguminosarum and Rhizobium etli is strikingly different when compared to that of enteric bacteria such as Escherichia coli. The Rhizobium species produce several unique enzymes that process the lipid A biosynthetic intermediate Kdo₂-lipid IV_A. These enzymes include a 1-phosphatase (LpxE), a 4´-phosphatase (LpxF), a 3-O-deacylase (PagL), and a lipid A oxidase (LpxQ). The biological functions and enzymological properties of many of the modification enzymes have remained unconfirmed and/or unknown. The purpose of these studies was to confirm the activities of these enzymes and to explore the functional significance of the resulting lipid A modifications.

To confirm the proposed biological functions of the enzymes in vivo, homologs of the lipid A phosphatases, LpxE and LpxF, from Francisella novicida and the lipid A oxidase LpxQ, were expressed heterologously in combination in E. coli. The resulting novel lipid A hybrids were analyzed by thin-layer chromatography (TLC) and electrospray ionization-mass spectrometry (ESI-MS).

The lipid A oxidase LpxQ, was characterized further biochemically. Two new purification procedures and a new in vitro assay were developed to analyze the properties of the enzyme. Purified LpxQ was shown to be dependent on oxygen and divalent cations for activity. Hydrogen peroxide was found to be a product of lipid A oxidation. A new fluorescence-based assay based on the detection of hydrogen peroxide was developed to monitor oxidation. LpxQ did not co-purifiy with any discernable cofactors, suggesting that it may employ a unique mechanism for the oxidation of lipid A.

The biological roles of LpxE and LpxF in plant nodulation were analyzed. Deletion mutants of the two phosphatases were generated in R. etli. The mutant strains accumulated the expected structures, confirming the specificity of the enzymes. Single and double phosphatase mutants were able to fix nitrogen in planta. Antimicrobial susceptibility testing indicated that dephosphorylation of lipid A increases resistance to cationic antimicrobials.

The biological role of the 3-O-deacylase, PagL, was also investigated. The pagL gene was identified using systematic homology searches. PagL was shown to be stimulated by calcium. A deletion mutant of the enzyme in R. etli was constructed and analyzed. The deletion mutant was found to be viable and unaltered in its ability to fix nitrogen. In conclusion, these studies have confirmed the roles of LpxE, LpxF, PagL, and LpxQ in Rhizobium lipid A biosynthesis and contributed new knowledge regarding the biochemical properties of LpxQ.

Bistable switches are commonly observed in the regulation of critical processes such as cell cycles and differentiation. The switches possess two fundamental properties: memory and bimodality. Once switched ON, the switches can remember their ON state despite a drastic drop in stimulus levels. Furthermore, at intermediate stimulus levels with cellular noise, the switches can cause a population to exhibit bimodal distribution of cell states. Till date, experimental studies have focused primarily on cellular mechanisms that generate bistable switches and their impact on cellular dynamics.

Here, I study emergent bistability due to bacterial interactions with either synthetic gene circuits or antibiotics. A synthetic gene circuit is often engineered by considering the host cell as an invariable "chassis". Circuit activation, however, may modulate host physiology, which in turn can drastically impact circuit behavior. I illustrate this point by a simple circuit consisting of mutant T7 RNA polymerase (T7 RNAP*) that activates its own expression in bacterium Escherichia coli. Although activation by the T7 RNAP* is noncooperative, the circuit caused bistable gene expression. This counterintuitive observation can be explained by growth retardation caused by circuit activation, which resulted in nonlinear dilution of T7 RNAP* in individual bacteria. Predictions made by models accounting for such effects were verified by further experimental measurements. The results reveal a novel mechanism of generating bistability and underscore the need to account for host physiology modulation when engineering gene circuits.

In the context of antibiotic treatment, I investigate bistability as the underlying mechanism of inoculum effect. The inoculum effect refers to the decreasing efficacy of an antibiotic with increasing bacterial density. Despite its implication for the design of antibiotic treatment strategies, its mechanism remains poorly understood. Here I show that, for antibiotics that target the core replication machinery, the inoculum effect can be explained by bistable bacterial growth. My results suggest that a critical requirement for this bistability is sufficiently fast turnover of the core machinery induced by the antibiotic via the heat shock response. I further show that antibiotics that exhibit the inoculum effect can cause a "band-pass" response of bacterial growth on the frequency of antibiotic treatment, whereby the treatment efficacy drastically diminishes at intermediate frequencies. The results have implications on optimal design of antibiotic treatment.

Lymph nodes are organs of efficiency. Once activated, they essentially function to optimize and accelerate the production of the adaptive immune response, which has the potential to determine survival of the host during an initial infection and protect against repeated infections, should specific and appropriate immunological memory be sufficiently induced. We now have an understanding of the fundamental structure of lymph nodes and many of the interactions that occur within them throughout this process. Yet, lymph nodes are dynamic and malleable organs and much remains to be investigated with regards to their responses to various types of challenges. In this work, we examined multiple inflammatory scenarios and sought to understand the complex ways that lymph nodes can be externally targeted to impact immunity. First, we outline a novel mechanism of cellular communication, where cytokine messages from the periphery are delivered to draining lymph nodes during inflammation. These signals are sent as particles, released by mast cells, and demonstrate the ability of the infected tissue to communicate to lymph nodes and shape their responses. Based on these interactions, we also explored the ability to therapeutically or prophylactically modulate lymph node function, using bioengineered particles based on mast cell granules, containing encapsulated cytokines. When we used these particles as a vaccine adjuvant, we were able to polarize adaptive immune responses, such as to promote a Th1 phenotype, or enhance a specific attribute of the immune response, such as the production of high avidity antibodies. We then explore three examples of lymph node-targeting pathogens: Salmonella typhimurium, Yersinia pestis and Dengue virus. Each of these pathogens has a well-characterized lifecycle including colonization of draining lymph node tissue. In the case of S. typhimurim, we report that the virulence this pathogen depends on a specific shut down of the chemotactic signals in the lymph node that are required to maintain appropriate cellular localization within it. Our results demonstrate that these architecture changes allow S. typhimurim to target the adaptive immune process in lymph nodes and contribute to its spread in vivo and lethality to the host. With Y. pestis, similar targeting of cellular trafficking pathways occurs through the modulation of chemokine expression. Y. pestis appears to use the host's cellular trafficking pathways to spread to lymph nodes in two distinct waves, first exploiting dendritic cell movement to lymph nodes and then enhancing monocyte chemoattractants to replicate within monocytes in draining lymph nodes. These processes also promote bacterial spread in vivo and we further demonstrate that blocking monocyte chemotaxis can prolong the host's survival. In the third example of pathogen challenge, we report for the first time that mast cells can contribute functionally to immunosurveillance for viral pathogen, here, promoting cellular trafficking of innate immune cells, including NK cells, and limiting the spread of virus to draining lymph nodes. For each of these three examples of lymph node targeting by microbial pathogens, we provide data that modulation of cellular trafficking to and within lymph nodes can drastically influence the nature of the adaptive immune response and, therefore, the appropriateness of that response for meeting a unique infectious challenge. Cumulatively this work highlights that a balance exists between host and pathogen-driven modulation of lymph nodes, a key aspect of which is movement of cells within and into this organ. Cytokine and chemokine pathways are an area of vulnerability for the host when faced with host-adapted pathogens, yet the lymph node's underlying plasticity and the observation that slight modulations can be beneficial or detrimental to immunity also suggests the targeting of these pathways with therapeutic intentions and during vaccine design.

Disease caused by the Gram-negative Haemophilus cryptic genospecies begins with colonization of the maternal genital or neonatal respiratory tract. The primary goal of this work was to identify and characterize the molecular determinant(s) of Haemophilus cryptic genospecies adherence as a means to better understand the specific adaptation of this species to the urogenital tract and neonatal respiratory tract. Using transposon mutagenesis of prototype strain 1595, we identified a locus that is essential for Haemophilus cryptic genospecies adherence to a variety of epithelial cell lines of both genital and respiratory origin. This locus encodes a protein called Cha that shares homology with trimeric autotransporters. Trimeric autotransporters are composed of an N-terminal signal peptide, an internal passenger domain that harbors adhesive activity, and a short C-terminal membrane anchor domain and are classically characterized by head-stalk-anchor domain architecture. By generating chimeric proteins, we demonstrated that the C-terminus of Cha trimerizes in the bacterial outer membrane and is capable presenting a heterologous passenger domain (Hia) in a functional form, thus confirming that Cha is a trimeric autotransporter. Southern analysis revealed that cha is unique to the Haemophilus cryptic genospecies and is ubiquitous among these strains.

Similar to a number of trimeric autotransporters, the passenger domain of Cha contains scattered clusters of YadA-like head domains associated with head-to-stalk neck adaptor motifs, predicted coiled-coil stalks and a series of identical tandem coding repeats which are not required for adherence. By evaluating the adherence capacity of H. influenzae expressing Cha deletion derivatives, we established that the N-terminal 473 residues of Cha harbor the binding domains responsible for Cha-mediated adherence to epithelial cells. In additional studies, we demonstrated that this same N-terminal region mediates bacterial aggregation through inter-bacterial Cha-Cha binding.

Further analysis revealed that variable Cha-mediated adherence is linked to spontaneous changes in the number of identical tandem repeats predicted to comprise a coiled-coil stalk domain. Variation in repeat copy number has a direct effect on Cha adhesive and aggregative activity, independent of an impact on transcription of the cha locus or surface localization of Cha protein. Moreover, length of Cha surface fibers correlates with repeat copy number expansion. We propose two hypotheses to explain how repeat expansion inhibits bacterial aggregation and host cell binding: 1) Variation in the number of 28-amino acid repeats may influence the conformation of Cha, thus changing the surface accessibility of the Cha binding pocket. 2) Repeat expansion results in the formation of long, flexible Cha fibers on the bacterial cell surface that may have a greater propensity to interact with neighboring Cha trimers at the N-terminus, thereby precluding adherence to other bacteria or host epithelial cells.

In additional studies screening adherent cryptic genospecies isolates for expression of Cha protein, we identified an additional, antigenically-divergent Cha variant that we refer to as Cha2. Amino acid sequence and domain comparison of Cha2 with Cha (now Cha1) revealed that the structurally undefined N-terminal sequences (encompassing the Cha1 adhesive and aggregative domain) are strikingly divergent. Inspite of this, Cha2 mediates efficient adherence to human epithelial cells, similar to Cha1.

Identification of Cha offers insight into the apparent tissue tropism associated with the Haemophilus cryptic genospecies. We speculate that the unique regulation of Cha adhesive activity enhances the adaptive capability of this pathogenic organism in the human host.

The Chlamydiaceae are Gram-negative, obligate intracellular bacteria that are significant pathogens of humans and animals. Intracellularly, the bacteria reside in a membrane-bound vacuole, called the inclusion, from which they manipulate host processes to create a niche optimal for survival and propagation. Acquisition of host-derived lipids is essential for chlamydial growth, yet the source of lipids and mechanisms of trafficking to the inclusion are not well-established. The inclusion avoids interaction with several classical membrane and lipid transport pathways. In a functional genomic screen to identify host modulating chlamydial proteins, our lab identified cytosolic lipid droplets (LDs) as potential target organelles of Chlamydia. LDs are postulated to function in many cellular processes, such as lipid metabolism and transport, membrane trafficking, and cell signaling; therefore, we hypothesized that LDs may be important for Chlamydia pathogenesis as a source of lipids or as a platform for regulating other cellular functions. Here, we characterize the interaction between eukaryotic LDs and the chlamydial inclusion.

We find that LDs are recruited to the Chlamydia inclusion, chlamydial infection disrupts neutral lipid homeostasis, and pharmacological prevention of LD formation inhibits chlamydial replication. Chlamydia produces proteins (Ldas) that localize with LDs in yeast and mammalian cells when transiently expressed and are exported out of the inclusion to peripheral lipid-rich structures during infection. By electron microscopy and live cell imaging, we observe the translocation of intact LDs into the Chlamydia inclusion lumen. Biochemical and microscopic analysis of LDs from infected cells reveals that LD translocation may occur at specialized subregions of the inclusion membrane. The Chlamydia Lda3 protein is implicated in LD tethering to the inclusion membrane, and displacement of the protective coat protein, ADRP, from LD surfaces. This phenomenon could provide access for lipases to the LD core for utilization by the replicating bacteria. Additionally, the functional domains of Lda3 involved in binding to LD and inclusion membranes are identified.

In these studies, we identify eukaryotic lipid droplets (LDs) as a novel target organelle important for Chlamydia pathogenesis and describe a unique mechanism of whole organelle sequestration not previously observed for bacterial pathogens. These results represent a fundamental shift in our understanding of host interactions with the chlamydial inclusion, and may represent a new area for research in the field of cellular microbiology.

Concurrent with the global escalation of the AIDS pandemic, cryptococcal infections are increasing and are of significant medical importance. Although improvements in antifungal therapy have advanced the treatment of cryptococcosis, the mortality rate is approximately 12% in medically advanced countries, and approaches 50% in less developed regions. Additionally, C. neoformans can cause infection in seemingly healthy individuals, elevating its status as a primary human pathogen. Although numerous studies have examined virulence properties, less is understood regarding host immune factors in the lungs during early stages of fungal infection. In the present thesis studies, I examined the roles played by pulmonary surfactant proteins in response to C. neoformans in vitro and in vivo. We demonstrate that SP-D, but not SP-A, binds to the yeast and increases phagocytosis of poorly encapsulated yeast cells by macrophages, yet concomitantly protects the pathogenic microbes from macrophage-mediated defense mechanisms. Furthermore, we show that SP-D functions as risk factor in vivo by protecting the yeast cells against oxidant species and thus facilitating disease progression. The results of these studies provide a new paradigm on the role played by surfactant protein D during host responses to C. neoformans and, consequently, impart insight into potential future treatment strategies for cryptococcosis.

Sexual reproduction in fungi is governed by a specialized genomic region called the mating-type locus (MAT). The ascomycetes, the largest phylum of fungi, primarily possess a bipolar mating system while the basidiomycetes, the second largest group, are mostly tetrapolar. The human fungal pathogen and basidiomycetous yeast Cryptococcus neoformans has evolved a bipolar mating system that encodes homeodomain (HD) and pheromone/receptor (P/R) genes. The MAT locus of C. neoformans is unusually large, spans greater than 100 kb, and encodes more than 20 genes. To understand how the pathogenic Cryptococcus species complex evolved this unique bipolar mating system, we investigated the evolution of MAT in closely and distantly related species and discovered an extant sexual cycle in Cryptococcus amylolentus.

Phylogenetic analysis using a six-gene multi-locus sequencing (MLS) approach identified the most closely related species to the pathogenic Cryptococcus species complex that are currently known. The two non-pathogenic sibling species, Tsuchiyaea wingfieldii and Cryptococcus amylolentus, and the more distantly related species Filobasidiella depauperata define the Filobasidiella clade. We also resolved the phylogeny of the species located in the sister clade, Kwoniella. A comprehensive tree dendrogram revealed that the 15 Tremellales species examined suggests a common saprobic ancestor. Moreover, the pathogenic Cryptococcus species have a saprobic origin but later emerged as pathogens. We further characterized the mating-type locus for T. wingfieldii and C. amylolentus by cloning and sequencing two unlinked genomic loci encoding the HD and P/R genes. Interestingly, linked and likely divergently transcribed homologs for SXI1 and SXI2 are present in T. wingfieldii and C. amylolentus, while the P/R alleles contain many genes also found in the MAT locus of the pathogenic Cryptococcus species. Also, hypothetical genes present in C. neoformans MAT are also MAT-linked in both species and indicate a possible translocation event between chromosomes 4 and 5 of C. neoformans. Our analysis of MAT in the sibling species indicates that T. wingfieldii is likely tetrapolar, and the C. amylolentus sequence comparison of the dimorphic SXI1 and SXI2 region and the pheromone receptor, STE3, suggests that C. amylolentus is also tetrapolar. The examination of MAT in these sibling species confirms the model for MAT evolution previously proposed in which this structure in C. neoformans and C. gattii evolved from an ancestral tetrapolar mating system. Moreover, the organization of MAT in these sibling species mirrors key aspects of the proposed intermediates in the evolution of MAT in the pathogenic Cryptococcus species, and for sex chromosomes in plants, animals, and alga in general.

We discovered an extant sexual cycle for C. amylolentus, a species previously thought to be asexual. Matings between two strains of opposite mating-types produce dikaryotic hyphae with fused clamp connections and uni- and bi-nucleate basidiospores. Genotyping of basidiospores using markers linked and unlinked to MAT revealed that genetic exchange (recombination) occurs during the sexual cycle of C. amylolentus, and it is likely that either aneuploids are generated during sex or more than one meiosis event occurs within each basidium. This is in contrast to C. neoformans, where only one meiotic event per basidium has been observed. Uniparental mitochondrial inheritance has also been observed in C. amylolentus progeny; similar to the pathogenic Cryptococcus species, mtDNA is inherited from the C. amylolentus MATa parent. Analysis of sex in C. amylolentus has provided insight into the mechanisms that phylogenetically related fungi employ in orchestrating sexual reproduction.

We also extended our analysis to include the distantly related tetrapolar basidiomycete Tremella mesenterica. We completed comparisons of MAT-specific genes between five strains of T. mesenterica and identified the regions that define its mating-type system. The HD locus is limited to the SXI1- and SXI2-like genes while the P/R locus is defined by STE3, STE12, STE20, and the pheromone gene, tremerogen a-13. Interestingly, many of the genes associated with the MAT locus of the pathogenic Cryptococcus species flank the HD and P/R locus and are not incorporated in MAT in T. mesenterica. The MAT region includes transposons and C. neoformans hypothetical genes also present in T. wingfieldii and C. amylolentus. The mating-type system in T. mesenterica reflects an ancestral intermediate in the evolution of the MAT locus in the pathogenic Cryptococcus species. In conclusion, this study provides an in-depth analysis on the structure, function, and evolution of an unusual mating-type locus with broader implications for the transitions in modes of sexual reproduction in fungi that impact gene flow in populations.

Hydrogen peroxide is used by animals and plants to deter the growth of microbial invaders by inflicting DNA lesions, protein oxidation and lipid membrane modifications. Pathogens protect themselves with enzymes and scavenging proteins. This study investigated population genetic, biochemical and genetic aspects of peroxide survival in Saccharomyces cerevisiae to address its importance for yeast biology and fungal pathogenicity.

Population genetic analyses of DNA sequences from five loci from 103 strains encompassing the known ecological spectrum of S. cerevisiae show that it is a recombining species with three divergent subgroups, which are associated with soil, fruit, and vineyards. Clinical isolates cluster with fruit isolates but are significantly more resistant to peroxide. Clinical isolates are genetically diverse, indicating multiple origins of the pathogenic lifestyle and eliminating the possibility that peroxide resistance is due to shared ancestry rather than it's importance for than its importance in colonizing the host.

Biochemical aspects of peroxide survival were studied in a resistant (high-survival) clinical isolate, a sensitive (low-survival) laboratory strain and their hybrid. Catalase activity and expression levels are indistinguishable among strains. Co-culture assays and growth curve records indicate that a secreted factor improves survival of the laboratory strain and that the phenotypic difference is most pronounced during exponential growth, excluding mechanisms of the General Stress Response effective during stationary phase. Semi-quantitative expression profiles of stress response candidate genes do not differ, suggesting a novel resistance mechanism.

To elucidate the genetic basis of peroxide survival, the hybrid was sporulated and 200 F1 segregants phenotyped and genotyped for oxidative stress candidate genes. Peroxide survival is a dominant quantitative trait and not linked to catalase, peroxidase or superoxide dismutase genes. 1,246 backcross segregants were phenotyped and 93 segregants selectively genotyped using microarrays. A 14-gene locus on chromosome XVI displayed marker-trait association. One gene, RDS2, encodes a zinc cluster protein acting as a regulator of drug sensitivity and contains a non-synonymous polymorphism whose exchange between the parental strains results a 15% decrease in survival in the clinical strain.

This work establishes a novel function for RDS2 in oxidative stress response and demonstrates the effect a quantitative trait nucleotide has on a clinically relevant phenotype.

The adenoviruses are nonenveloped double stranded DNA viruses, which cause enteric dysentary and respiratory infection. Adenovirus has become a focus of the water treatment community because of its apparent resistance to ultraviolet disinfection; it is the basis for stringent new EPA regulations regarding all viruses in both surface and ground waters. Most of the work done so far, however, has involved the use of monochromatic (254 nm) low pressure (LP) UV sources and subsequent assay of viral infectivity in cell culture models. LP UV lamps primarily damage DNA, while polychromatic UV sources may damage other parts of the virus as well. Recent research has shown that these newer, polychromatic UV sources--such as medium pressure (MP) UV--are more effective than monochromatic LP UV for disinfection of adenovirus. The objectives of this work were to study adenoviral response to UV using both LP and MP UV as well as using both standard cell culture infectivity assays and more direct methods of assessment based on molecular biology. These include quantitative long PCR for assessment of DNA damage and SDS-PAGE for assessment of protein damage; transmission electron microscopy was used to examine the structure of UV treated viral particles. This work was only the second significant study to show the response of adenoviruses to medium pressure UV and the first to thoroughly examine the response of adenoviruses to both LP and MP UV using cell culture-independent methods. Results confirm that adenovirus is sensitive to MP UV when assayed in cell culture; they show that LP and MP UV are equally effective at inducing damage to the adenoviral genome and that MP UV is more effective than LP UV at damaging the viral proteins. This work helps deepen our understanding of UV disinfection of adenovirus.

The relatively simple innate immune system of Caenorhabditis elegans and the number of traits that facilitate genetic and genomic analysis using this organism have nurtured rapid advances into the understanding of C. elegans innate immunity during the last few years. However, traditional methods of isolating and mapping C. elegans mutants exhibiting aberrant immune responses to pathogen infection are often labor intensive and time consuming. Therefore, a simple and rapid means of isolating and mapping C. elegans immune mutants will increase the number of mutants that can be studied. Salmonella enterica, as well as other bacterial pathogens, has been described to cause a significant distension of the C. elegans intestinal lumen, which correlates with death of the nematode. C. elegans mutants which exhibit a weakened immune response would therefore be expected to develop intestinal distension at an earlier time point than wild type. Likewise, mutants which exhibit an enhanced immune response would be expected to develop intestinal distension at a later time point than wild type. Taking advantage of this correlation, we designed a novel approach to isolating C. elegans mutants which exhibit aberrant immune responses to the bacterial pathogen, S. enterica. Furthermore, we validated and optimized the use of Amplifluor®, a high-throughput genotyping system, for use in C. elegans single nucleotide polymorphism (SNP) mapping.

To date, the only known negative regulators of innate immunity in C. elegans are dependent on the FOXO transcription factor, DAF-16 and regulate lifespan in addition to immunity. Therefore, we focused our efforts on identifying additional negative regulators of innate immunity by screening for mutants which display a reduced accumulation of S. enterica at a time point when wild-type nematodes are packed with bacteria. In a genetic screen for C. elegans mutants which display reduced accumulation of S. enterica/GFP, we identified a mutation in nol-6, a nucleolar protein containing a nucleolar RNA-associated protein (Nrap) domain which is conserved across eukaryotic organisms. nol-6 is implicated in ribosomal RNA (rRNA) processing during the early stages of ribosome biogenesis. We show that knockdown of nol-6 as well as other nucleolar genes leads to a reduction of pathogen accumulation and enhanced resistance to killing by pathogen. In addition, we demonstrate that enhanced resistance is dependent on p53/cep-1. Furthermore, microarray analysis shows a significant enrichment of upregulated genes that have previously been shown to be dependent on p53/cep-1 for induction following ultraviolet radiation. These results represent the first evidence that C. elegans innate immunity is regulated by the nucleolus through a p53/cep-1-dependent mechanism.

Transcription regulators are proteins that bind to specific DNA sequences in order to control the expression of specific genes. Often the sequences that are bound are not identical, but contain deviations from a common "consensus" sequence. The proteins that recognize these non-consensus sites must be able to recognize a variety of related sequences. MetJ is the transcription regulator that controls the expression of genes involved in methionine biosynthesis and transport in E. coli and other related organisms. A consensus sequence is known, but almost all the naturally occurring binding sites for MetJ differ from this. The goal of this dissertation is to understand how MetJ recognizes its various target sites within the context of the genomic DNA in which they are embedded. This work uses a variety of biochemical and biophysical techniques to further our understanding of an important regulatory protein.

Chapter 2 describes the results of both in cell and in vitro NMR showing that MetJ associates with non-specific genomic DNA in the cell, and that specific DNA (containing the consensus sequence) can successfully compete with a large excess of non-specific DNA for MetJ binding.

Chapter 3 describes work performed with small-angle neutron scattering showing different modes of MetJ binding to DNA of variable length and sequence.

Chapter 4 extends the neutron-scattering results by using analytical ultracentrifugation to look at MetJ binding to a wide variety of DNA sequences, both in the presence and absence of its co-factor, S- adenosylmethionine (SAM). Evidence is presented for SAM-mediated binding to both specific and non-specific DNA, as well as the importance of cooperativity in binding multiple MetJ molecules to a single DNA.

Large amounts of human pharmaceutical products are consumed worldwide. Many drugs and their metabolites, referred to as pharmaceutically active compounds (PhACs), are not fully metabolized prior to household discharge resulting in their common occurrence in wastewater treatment plants (WWTPs). In most instances, WWTPs present the first treatment opportunity for removing PhACs and preventing significant environmental exposure. Because most municipal WWTPs rely on the microbial component of the activated sludge process, there is a need to estimate the influence of PhACs in wastewater influent on the activated sludge microbial communities and the treatment performance of WWTPs. The objective of this dissertation was to determine the impact of selected PhACs (i.e., ketoprofen, naproxen, clofibric acid, carbamazepine and gemfibrozil) on activated sludge microorganisms and key individual microbial species in domestic wastewater treatment. Analyses were performed in batch reactors initially and then in laboratory-scale sequencing batch reactors (SBR) which mimic WWTP operations. Ammonia oxidizing bacteria (AOB) were selected as indicator organisms because of their importance in wastewater treatment and demonstrated sensitiveness to toxic compounds.

The batch experiments results suggested that microbial growth inhibition was correlated to organic loadings. In the presence of 0.2% (v/v) ethanol, significant inhibition, ranging from 34 to 43%, was observed for all PhACs other than clofibric acid.

Nitrification inhibition studies using Nitrosomonas europaea, a model AOB strain showed that ketoprofen, naproxen, carbamazepine and gemfibrozil inhibited nitrite production. The corresponding maximum nitrification inhibition rates were 25, 29, 22 and 26%, respectively. Inhibition was shown to increase with PhAC concentration for concentrations greater than 0.1 µM. Results from membrane integrity tests suggest that the inhibition may be due to the disturbance of the cell membrane by PhACs and such inhibition was shown to be irreversible.

Even though PhACs were shown to inhibit the nitrification rate in pure culture studies, the performance of SBRs exposed to individual PhACs was not adversely affected neither in terms of COD nor ammonia removal. Microbial fingerprinting for both total bacteria and AOB confirmed that no significant shifts occurred when microbial communities were exposed to PhACs. However, some PhACs introduced in binary mixture were found to both inhibit the nitrification of N. europaea as well as the performance of SBRs. The mixture composed of 0.5 μM ketoprofen and 0.5 μM naproxen showed significant inhibition (25%) on the nitrite production of N. europaea although neither 0.5 μM ketoprofen nor 0.5 μM naproxen had significant effect when presented alone. Similarly, both COD and ammonia removal were significantly impacted by binary mixtures of PhACs. These results suggest that mixture effects can play an important role in an overall treatment's nitrification potential and this phenomenon should be further investigated.

The Piedmont region of the southeastern United States has undergone considerable land-use change since settlement by Europeans and Africans. Forests were cleared for agriculture, followed centuries later by land abandonment. Following abandonment, natural recruitment, plantings for erosion control, and plantation forestry have resulted in a large area of the region covered by loblolly pine, Pinus taeda. Today, the Piedmont is a mosaic of farm fields, pastures, pine forests, and relic woodlots. The Calhoun Experimental Forest, located in Union County, SC, has provided a unique history of land use change's alteration of soil properties and processes, the ability of reforestation to restore or deplete soil fertility, and provided insights into the effects this change has on biological diversity.

In this work, the diversity of fungi living in soil is examined in the context of land-use change and soil biogeochemical change in and around the Calhoun Forest. This study uses molecular tools to identify fungal species from soil and to identify mycorrhizal associates of loblolly pine in a bioassay of propagule diversity, and proposes a novel use of quantitative PCR to quantify the relative abundance of major fungal families affected by land-use change.

Fungal diversity in soils is high in all land uses, but fungal communities shift from agricultural field communities largely comprised of unicellular ascomycetes and basal lineages to forest communities dominated by saprophytic and symbiotic basidiomycetes. In addition to this shift across a land use gradient, fungal communities are also responding to changes in carbon quantity and quality, biologically available nitrogen and phosphorus, pH, acidity and texture.

ECM propagule communities also differ across a land use gradient of cultivated fields, grasslands, pine forests, and mixed hardwood stands. There are few ECM propagules able to associate with loblolly pine in cultivated and grassland soils. There is a trend towards higher ECM diversity in the hardwood and pine soils, and both of those soil communities are distinct from each other as well as from soils from field treatments.

Quantitative PCR, coupled with a nested set of taxon-specific, fungal primers, is a potential way to estimate the abundance of the given taxon relative to all fungi in an environmental DNA. Primers specific to several taxonomic level of fungi were tested to confirm amplification in PCR, then were tested for taxonomic specificity by generating clone libraries with environmental DNA. Several of the successful primers were tested with soil DNA extracts in QPCR and the calculated ratios of fungal abundance varied widely by method of analysis. The results suggest that many repeated measurements and many replicates are required for a robust estimate of the relative abundance of a specific taxon.

TThe proposed research will provide a set of 16 near complete mtDNA gene orders. The observed gene rearrangements will be used to investigate the phylogeny of the Sub-Class Hydroidolina and the Family Hydractiniidae. All the medusozoan classes contain a linear mitochondrial genome (mtDNA genome), whereas the Class Anthozoa contains a circular mtDNA genome (Bridge et al., 1992). The linear structure of the medusozoan mtDNA genomes is the most likely reason why these genomes are underrepresented, because the most rapid methods of mtDNA genome sequencing take advantage of the circular nature of most animal mtDNA. In a circular genome where mtDNA gene order is unknown prior to isolation, the forward and reverse primer(s) are designed based on the sequences of one or two highly conserved regions. Linear mtDNA requires a more involved approach, making it more difficult to sequence in its entirety than circular mtDNAs. In chapter 1, a novel assay to determine linear mtDNA gene orders is presented. In chapter 2, the near-complete mtDNA genome sequences are presented, as well as 7 near-complete mtDNA gene orders determined by the mtDNA gene order assay. These data are used to investigate relationships in the Family Hydractiniidae. Finally, in chapter 3, an additional 9 near complete mtDNA gene orders are used to explore relationships in the sub-class Hydroidolina. This study significantly increases the number of known near-complete mtDNA genomes, as well as their mtDNA.

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of morbidity and mortality worldwide. The causative agent of traveler's diarrhea, ETEC is often associated with cholera-like disease, especially in developing countries. One major virulence factor released by ETEC is the heat-labile enterotoxin LT, which upsets the balance of electrolytes in the intestine. LT is highly similar to cholera toxin (CT) produced by Vibrio cholerae, both in structure and function. The toxin consists of a single catalytically active A subunit and a ring of five B subunits mediating its binding and secretion. Previous work from our lab has shown that, after export by the type II secretion (T2S) system, LT associates with lipopolysaccharide (LPS) on the bacterial surface. However, little is known about what identifies LT as a T2S substrate, and the portion of the toxin that mediates LPS binding has not previously been defined. Site-directed mutagenesis of residues in a peripheral sugar binding pocket of the toxin was performed, revealing mutations that affect its binding to LPS, as determined by an in vitro cell surface binding assay. One binding mutant, which is expressed and secreted at wild-type levels from ETEC, holds particular promise for further studies of the role of the LT-LPS interaction. Interestingly, some mutations made affected the secretion of the toxin as detected by ganglioside-binding ELISAs of cell-free supernatant, and several mutations affected both secretion and LPS binding. These mutations identify residues of the toxin that are involved in its secretion and association with LPS. In addition, we introduced mutations affecting the secretion of LT into CT, due to the high similarity between the two toxins. While one mutation affects the secretion of each, other mutations affect one toxin but not the other. These results demonstrate that LT and CT are recognized in different ways during T2S. Combined with an analysis of the effects of secretion mutations on the stability of the toxin, the results described here highlight the delicate balance between structure and function of the LT B subunit.

Fungi have a genetically controlled sex determination system, which is governed by a small, sex-specific region in the genome called the mating-type locus (MAT). In the basidiomycetous yeast Cryptococcus neoformans, the pathogen that causes cryptococcal meningitis and cryptococcosis, sex has been associated with virulence. To further understand how sex is genetically regulated in C. neoformans, we focused our studies on the evolution of the MAT locus and molecular dissection of the pheromone signaling pathway that controls sexual development. Two MAT-linked meiotic recombination hotspots that likely drove the assembly and rearrangement of MAT were identified. Fine mapping through the integration of genetic markers established that two hotspots, one on each side of the MAT locus, are located in an ~10 kb and ~5 kb region. Plotting the G + C content along MAT and the flanking regions revealed a strong association between the location of these two hotspots and a high G + C content. By deletion and insertion of the G + C rich region, we demonstrated that the high G + C rich region is required but not sufficient to induce recombination. On the other hand, to provide direct experimental evidence to support the previously proposed model for the evolution of MAT, we sought to recapitulate the ancestral tetrapolar, and the intermediate tripolar mating systems of C. neoformans by manipulating the MAT structure to model a tetrapolar system. In the two modified "a" and "α" strains, the sex-determining genes SXI1α or SXI2a residing at the MAT locus were disrupted and the wild-type allele of these two genes was then reintroduced at another genomic location (URA5) that is unlinked to MAT. Our results show that C. neoformans can complete the sexual cycle with a tetrapolar mating configuration and the transitional tripolar state might be under strong negative selection pressure, which could have facilitated the transition from a tripolar state to the final bipolar mating system.

The MAT locus is the major determinant of the sexual identity of a cell, but several signaling pathways, including the pheromone signaling pathway, are required to regulate mating and sexual development. Many components of the pheromone signaling pathway have been identified; however, it is less clear what lies upstream of the MAPK cascade. To address this question, we studied the role of two Gα subunits (Gpa2, Gpa3) in mating and concluded that they share both redundant and divergent roles in mating. gpa2 gpa3 double mutants, but neither gpa2 nor gpa3 single mutants, are sterile in bilateral crosses. In their GTP-bound form, they signal in opposition: Gpa2 promotes mating whereas Gpa3 inhibits. Furthermore, we also studied the functions of a novel upstream component Cpr2, a pheromone receptor-like gene, in pheromone signaling and sexual development. All lines of evidence suggest that Cpr2 is a constitutive ligand-independent receptor that, when expressed, engages the same G-proteins and activates the same pheromone signaling pathway as the canonical ligand-activated pheromone receptors. Expression of Cpr2 is induced post cell fusion during mating, and likely introduces a positive feedback loop to allow a self-perpetuating signaling state to enable efficient mating. Cells lacking this receptor are fertile, but produce abnormal filamentous structures. Overexpression of CPR2 in a or α cells strongly enhances fruiting, an alternative same-sex mating process in C. neoformans. Therefore, Cpr2 establishes a new paradigm for a naturally occurring constitutively active GPCR that governs cell fate in fungi.

Finally, we described a sex-induced silencing (SIS) phenomenon in C. neoformans. Using genetic approaches, we showed that SIS is triggered by a tandem insertion of a transgene during the sexual cycle. Interestingly, only a proportion of progeny carrying the transgene are silenced. Gene deletion, RIP, or DNA methylation do not contribute to SIS but the RNAi machinery is required. In conclusion, these studies provide further understanding of sex in C. neoformans from different perspectives, which invites comparisons to other fungal and even more broadly, eukaryotic pathogens to address the role of sex in evolution.

Although the majority of the population carries Candida spp as normal components of their microflora, these species are important human pathogens that have the ability to cause disease under conditions of immunosuppression or altered host defenses. The spectrum of disease caused by these species ranges from cutaneous infections of the skin, mouth, esophagus and vagina, to life-threatening systemic disease. Despite increases in drug resistance, the antifungal armamentarium has changed little over the past decade. Thus increasing our understanding of the life cycles of these organisms, not only how they propagate themselves, but also how genetic diversity is created within the population is of considerable import. Additionally expanding our knowledge of key signal transduction cascades that are important for cell survival and response to stress will add in developing new antifungal therapies and strategies.

This thesis addresses both of these key areas of fungal pathogenesis. In the first chapter, we use genome comparisons between parasexual, asexual, and sexual species of pathogenic Candida as a first approximation to answer the question of whether examining genome content alone can allow us to understand why species have a particular life cycle. We start by examining the structure of the mating type locus (MAT) of two sexual species C. lusitaniae and C. guilliermondii. Interestingly, both species are missing either one or two (respectively) canonical transcription factors suggesting that the control of sexual identity and meiosis in these organisms has been significantly rewired. Mutant analysis of the retained transcription factors is used to understand how sexual identity and sporulation are controlled in these strains. Secondly, based on the observation that these species are missing many key genes involved in mating and meiosis, we use meiotic mapping, SPO11 mutant analysis, and comparative genome hybridization to demonstrate that these species are indeed meiotic, but that the meiosis that occurs is occasional unfaithful generating aneuploid and diploid progeny.

In the second and third chapters we examine the calcineurin signaling pathway, which is crucial for mediated tolerance to cellular stresses including cations, azole antifungals, and passage through the host bloodstream. First, we show that clinical use of calcineurin inhibitors in combination with azole antifungals does not result in resistance to the combination, suggesting that if non-immunosuppressive analogs could be further developed this combinatorial strategy may have great clinical efficacy. Second, we use previous studies of the calcineurin signaling pathway in S. cerevisiae to direct a candidate gene approach for elucidating other components of this pathway in C. albicans. Specifically, we identify homologs of the RCN1, MID1, and CCH1 genes, and use a combination of phenotypic assays and heterologous expression studies to understand the roles of these proteins in C. albicans. Although the mutant strains share some phenotypic properties with calcineurin deletion strains, none completely recapitulate a calcineurin mutant.

In the last chapter, we examine the plausibility of targeting the homoserine dehyrogenase (Hom6) protein in C. albicans and C. glabrata as a novel antifungal strategy. Studies in S. cerevisiae had demonstrated a synthetic lethality between hom6 and fpr1, the gene encoding FKBP12 a prolyl-isomerase that is the binding target of the immunosuppressant FK506. Thiss synthetic lethality was due to the buildup of a toxic intermediate in the methionine and threonine biosynthetic pathway as a result of deletion of hom6 and inhibition of FKBP12. We deleted HOM6 from both C. albicans and the more highly drug-resistant species C. glabrata. Studies suggest that regulation of the threonine and methionine biosynthetic pathway in C. albicans has been rewired such that the synthetic lethality between hom6 and FKBP12 inhibition no longer exists. However, in C. glabrata preliminary analysis suggest that similarly to S. cerevisiae hom6 and inhibition of FKBP12 can result in cell death.

The Gram-negative bacteria Chlamydia trachomatis is a common sexually transmitted pathogen that can cause severe sequelae including cause pelvic inflammatory disease and sterility. This obligate intracellular pathogen effectively manipulates host cellular functions by secreting virulence factors across its membrane bound vacuole. Identifying these virulence components and how they help in establishing an environment conducive for bacterial growth is central to understanding chlamydial pathogenesis. This is experimentally challenging due to a lack of tools to perform molecular genetic studies. In the absence of genetic tools, we developed a yeast model system to identify and characterize chlamydial proteins involved in virulence mechanisms. In this study we describe the identification of twenty-eight proteins potentially involved in modulating host cellular functions and the secretion of virulence factors into the host. Since the delivery of virulence proteins by a type three secretion (T3S) system is a critical step for Chlamydia, we identified the proteins that interacted with the T3S apparatus by yeast two-hybrid analysis. We discovered several novel interactions between and determined that the C. trachomatis T3S apparatus displayed a similar architecture to that of other T3S systems. Furthermore with these approaches we identified networks of proteins that interacted with the secretion apparatus including a novel secretion chaperone protein. We characterized Ct260/Mcsc one of the putative secretion and demonstrated that it represents a novel class 1B secretion chaperone protein. Unlike other known chaperones, Mcsc directly interact with a conserved component of the T3S apparatus cytoplasmic domain, CdsQ. These finding represents a novel mechanism by which the secretion chaperone protein Ct260 may increase the secretion efficiency of its effector cargo and may reveal new facets of secretory cargo recognition by T3S systems.

Afforestation, the conversion of historically treeless areas into forests, is a rapidly spreading land-use change with the potential to sequester carbon. Afforested plantations typically feature fast growing exotic tree species that give landowners rapid returns. The efficient growth of plantations compared to less intensively managed forests also can provide greater timber yields in a smaller area. This increased efficiency in turn could require fewer acres to meet global forest product demands and could also reduce the need to log intact primary forests. Reduced primary forest harvest and high primary productivity make afforestation a highly efficient carbon sequestration tool.

However, the rapid growth and planting disturbance due to afforestation can have deleterious effects on soils and hydrology that undermine its benefits in some locations. The effects on hydrology include depletion of groundwater and reduced or complete elimination of surface water flow. Additionally, groundwater use can lead to increased concentrations of salts and trace metals in soil that could be deleterious for future plant productivity. Plantations have also been shown to acidify surface soils and stream water and to reduce soil carbon and nitrogen.

Despite the known effects of afforestation on soils, there has been little research on the mechanisms controlling these effects. For instance, there have been few studies on the effects of afforestation on soil microbes which mediate most biogeochemical processes. There is also little knowledge on what controls the effects of afforestation on soil carbon and nitrogen, vital indexes of soil quality, across regions with high levels of afforestation. The overarching goal of this dissertation is to examine the effects of afforestation on soils, microbes, and biogeochemical processes across local, regional and global scales. Understanding the mechanisms by which afforestation alters soils and biogeochemical cycling and how these mechanisms change across different scales will aid in evaluating the true costs and benefits of afforestation. These results will be useful in determining if the benefits of afforestation will continue to outweigh its costs in the long-term.

The goal of Chapter 1 is to evaluate how afforestation across the globe affects mineral soil quality, including pH, sodium, exchangeable cations, organic carbon, and nitrogen, and to examine the magnitude of these changes in regions where afforestation rates are high. To control for different initial soil conditions across the globe, I examined paired sites of afforested plantations and controls. Controls included land-use types that are frequently afforested, such as grasslands, shrublands, and pastures. I also examined potential mechanisms to reduce the impacts of afforestation on soils and to maintain long-term productivity. Across diverse plantation types (153 sites) to a depth of 30cm of mineral soil, I observed significant decreases in nutrient cations (Ca, K, Mg), increases in sodium (Na), or both with afforestation. For the global dataset, afforestation reduced soil concentrations of the macronutrient Ca by 29% on average compared with native controls (p<0.05). Afforestation by Pinus alone decreased soil K by 23% (p<0.05). Overall, plantations of all genera also led to an average 71% increase of soil Na (p<0.05). Average pH decreased 0.3 units (p<0.05) with afforestation. Afforestation caused a 6.7% and 15% (p<0.05) decrease in soil C and N content respectively, though the effect was driven principally by Pinus plantations (15% and 20% decrease, p<0.05). Carbon to nitrogen ratios in soils under plantations were 5.7-11.6% higher (p<0.05). The major implication of these results are that in several regions with high rates of afforestation, cumulative losses of C, N, Ca, and Mg are likely in the range of tens of millions of metric tons. The decreases indicate that trees take up considerable amounts of nutrients from soils; harvesting this biomass repeatedly could impair long-term soil fertility and productivity in some locations. Based on this study and a review of other literature, I suggest that proper site preparation and sustainable harvest practices, such as avoiding the removal or burning of harvest residue, could minimize the impact of afforestation on soils. These sustainable practices could in turn slow erosion, organic matter loss, and soil compaction from harvesting equipment, maintaining soil fertility to the greatest extent possible.

Soil microbes are highly diverse and control most soil biogeochemical reactions. Given the observed changes in Chapter 1, in Chapters 2 and 3 I examined how microbial functional genes and biogeochemical pools responded to the altered chemical inputs accompanying afforestation. I examined paired native grasslands and adjacent Eucalyptus plantations (previously grasslands) in Uruguay, a region that lacked forests before European settlement. Along with measurements of soil carbon, nitrogen, and bacterial diversity, I analyzed functional genes using the GeoChip 2.0 microarray that simultaneously quantified several thousand genes involved in soil carbon and nitrogen cycling. Plantations and grasslands differed significantly in functional gene profiles, bacterial diversity, and biogeochemical pool sizes. Afforestation decreased both bacterial diversity and richness compared to grasslands, though diversity remained relatively high. Most grassland functional gene profiles were similar, but plantation profiles generally differed from grasslands due to differences in functional gene abundance across many microbial groups. Eucalypts decreased ammonification and N-fixation functional genes by 11% and 7.9% (p<0.01) which correlated with decreased microbial biomass N and more NH4+ in plantation soils. Chitinase, an important carbon polymer degrading enzyme, decreased in functional gene abundance 7.8% in plantations compared to grasslands (p=0.017), and C polymer degrading genes decreased by 1.5% overall (p<0.05), which likely contributed to 54% (p<0.05) more C in undecomposed extractable soil pools and 27% less microbial C (p<0.01) in plantation soils. In general, afforestation altered the abundance of many microbial functional genes corresponding with changes in soil biogeochemistry. These changes were driven by shifts in the whole community functional gene profile, not just one or two constituent microbial taxa. Such changes in microbial functional genes correspond with altered C and N storage and have implications for long-term productivity in these soils.

The area studied in Chapters 2 and 3 lies near the middle of a precipitation gradient that stretches across the Rio de la Plata grasslands. In Chapter 4 I studied if the effects of afforestation on soil C and N from Chapters 2 and 3 varied with different precipitation levels. The effect of afforestation on soil C has been shown to depend on mean annual precipitation (MAP), with drier sites gaining C and wetter sites losing C with afforestation. This precipitation dependence of soil C changes with afforestation may be controlled by changes in soil nitrogen (N) cycling. In particular, loss of N due to leaching after afforestation could lead to soil C losses. However, the link between C and N changes due to afforestation has primarily been suggested by models and to my knowledge has never been explicitly tested across a precipitation gradient. The goal of this study was to test how precipitation affects changes in labile and bulk pools of soil C and N across a precipitation gradient, which will provide novel insight into the linkage between land-use change, different pools of soil C and N, and precipitation. I conducted this study across a gradient of precipitation in the Rio de la Plata grasslands of Argentina and Uruguay which ranged from 600mm to 1500mm of precipitation per year. The sites were all former grasslands that had been planted with Eucalyptus. I found that changes in bulk soil C and N were related to MAP with drier sites gaining and wetter sites losing C and N (R2=0.59, p=0.003), which supports the idea that N losses are strongly linked to C losses with afforestation. C and N in microbial biomass and extractable pools followed similar patterns to bulk soil C and N. Interestingly, losses of C and N decreased as the plantations aged, suggesting that longer rotation times for plantations could reduce potential soil carbon and nitrogen losses. These results indicate that afforestation is still be a valuable tool for carbon sequestration, but calculations of the benefits of afforestation must take into account site factors such as age and precipitation to accurately calculate total sequestration benefit and ensure continued high productivity and carbon sequestration.

In conclusion, afforestation could be an effective tool for carbon sequestration. However, its benefits need to be carefully weighed against its costs for soil such as reduced microbial diversity, decreased soil microbial functional capacity, losses of soil organic matter, and nutrient depletion. Careful management and consideration of afforestation is needed to ensure the greatest benefits with the least long-term damage to soils.