Neurofibromatosis type 1 (NF1) is an inherited disease characterized by the appearance of multiple neurofibromas and an increased incidence of malignant schwannomas, both of which contain hyperproliferative Schwann cells. Our laboratory previously reported that Schwann cells produce stem cell factor (SCF), a multi potential growth factor known to be involved in mast cell migration and growth. Given the fact that mast cell numbers are increased in both neurofibromas and malignant schwannomas, we set out to evaluate a potential role for SCF in the development of NF1 lesions. First we studied the effects of high doses of recombinant SCF on mast cell numbers in vivo, and found that dermal and peritoneal mast cell numbers were decreased. Next, we examined the expression of stem cell factor and its receptor, Kit, in neurofibromas and malignant schwannoma tumors and cell lines. Using an RNase protection assay, we find that each of four human malignant schwannoma cell lines express only the membrane-bound isoform of SCF messenger RNA. In contrast, neurofibroma, vestibular schwannoma, and acoustic neuroma tissues, as well as the majority of human fibroblast sources, all express the soluble form. Low level expression of Kit protein was detected on all four malignant schwannoma cell lines. However, Kit expression by Schwann cells was not indicated by immunohistochemical analyses of neurofibroma and malignant schwannoma sections, although Kit was readily detected on the mast cells within these lesions. We report that bone marrow-derived mouse mast cells appear to be driven toward a connective tissue phenotype when cultured in the presence of conditioned media from Schwann cells of wild type and NF1 +/- knockout mice. This effect was not observed in cultures containing conditioned medium from Schwann cells of NF1 +/- knockouts. In addition, the latter appeared to augment the proliferation of mast cells in response to exogenous cytokines. Together, these results suggest a significant role for stem cell factor and Kit in the lesions of NF1. In addition, functional neurofibromin, the product of the NF1 gene, may be required for proper regulation of SCF isoform expression. The mechanisms by which this expression is regulated remain to be clearly defined.

The genus Naegleria is composed of a distinct group of free-living amoeboflagellates that include both pathogenic and nonpathogenic species. N. fowleri, the only pathogenic species of Naegleria to be isolated from humans, is the etiological agent of primary amoebic meningoencephalitis, a rare but rapidly fatal disease of the central nervous system in humans and in laboratory animals. The mechanisms of pathogenicity and the determinants of virulence of N. fowleri are unknown. Both pathogenic and nonpathogenic Naegleria activate the alternative complement pathway, however pathogenic N. fowleri are complement-resistant and nonpathogenic N. gruberi are complement-sensitive. The ability to resist complement-mediated lysis may be an important determinant of virulence of N. fowleri. These studies demonstrate that pathogenic N. fowleri possess at least two mechanisms for resisting complement lysis. Pathogenic N. fowleri synthesize a surface associated protein which appears to possess structural as well as functional homology to the human complement regulatory glycoprotein, CD59. Also, other surface glycoproteins appear to play a role in regulating complement lysis either directly or by indirect inhibitory mechanisms. In addition to complement regulatory glycoproteins, pathogenic N. fowleri possess the ability to remove membrane deposited complement proteins, C5b-C9, from their cell surface by membrane vesiculation. The presence of complement regulatory proteins and the ability to vesiculate in response to serum complement, alone or in combination, serves to protect pathogenic N. fowleri from complement-mediated damage. Nonpathogenic N. gruberi do not appear to possess surface complement regulatory proteins or the ability to vesiculate in response to serum complement. Additional studies demonstrate that growth medium modulates complement resistance and virulence. More importantly, by using changes in growth medium, an in vitro model was developed for differentially expressing proteins associated with the complement-resistant state. The induction of these de novo synthesized proteins may serve as markers of virulence and complement resistance in pathogenic N. fowleri amoebae.

Organisms of the genus Naegleria differentiate from feeding, dividing amebae into temporary swimming flagellates when deprived of nutrients. Factors critical for enflagellation of the pathogenic species, N. fowleri, were delineated and optimal conditions established for evoking reproducible conversion of N.fowleri populations. N. fowleri enflagellation differed from that of N. gruberi in its dependence upon growth phase, population density during enflagellation and in the timing and extent of the conversion. N. fowleri amebae from stationary phase cultures grown at 37°C, washed free of medium and suspended in nutrient~free ameba saline, acquired flagella and converted to mature flagellates in a synchronous manner beginning 90 minutes after subculture to ameba saline. No prolonged intermediate round phase occurred during N. fowleri enflagellation such as has been seen with N. gruberi. Enflagellation of N. fowleri was prevented by actinomycin D and cycloheximide added at the time of subculture to non-nutrient medium. Delayed additions of the same inhibitors caused hastened reversion of flagellates, suggesting a requirement for continued synthesis. Ultrastructural changes during N. fowleri enflagellation generally paralleled those observed in E. gruberi except that flagellum outgrowth in the former occurred on cells while still ameboid. An extensive complement of cell polypeptides continued to be synthesized at a reduced level during enflagellation; no limited number of species was made in exceptional abundance. A moderate number of qualitative and quantitative changes were detected in the amounts of individual polypeptides resolved by two-dimensional electrophoresis when amebae enflagellated, some of which also occurred in a non-enflagellating g, fowleri strain and may therefore be related to starvation. Quantitative, computer-assisted densitometric analyses of polypeptide autoradiograms revealed that larger and more acidic proteins were relatively more abundant in amebae than in flagellates and that a correlation existed between molecular size and charge of N. fowleri polypeptides. Several observations in both E. fowleri and N. gruberi enflagellation implicate regulatory mechanisms in addition to, or coordinated with, de novo protein synthesis. Enflagellation of Naegleria species provides a useful paradigm in which to study the contributions of various regulatory processes upon the expression of a differentiated state in an eukaryotic cell.

The nature and evolution of antibiotic resistance in pathogens and the appearance of pandemics such as HIV emphasize the importance of considering a variety of novel approaches to develop alternative vaccine methods and useful new components. The goal of this research was to generate and test an innovative, cost effective antigen display and delivery system that uses micro particulate gas vesicles. These unique proteinaceous organelles are naturally produced by halophilic archaea and their biogenesis is inherently controlled. Preliminary evidences indicate they can be genetically manipulated to display peptides coded by inserts of exogenous pathogen DNA and specified sequences can be verified and tested in vivo. Such characteristics are desirable in an antigen presenting system and potentially important for applications in vaccine development. The non-toxic Halobacteria sp. should be acceptable for use in oral as well as parenteral delivery. A highly organized surface of the Gv can be used for antigen presentation and these stable protein structures have an intrinsic adjuvant activity. The results indicate that a functional display/delivery system can be derived using recombinant plasmids consisting of DNA from the simian immunodeficiency virus infecting the sooty mangabeys [SIVsm] and DNA of the gene cluster [ gvp] encoding gas vesicle proteins. Therefore it was possible to: (1) establish a system for generating a multiepitope display library [MED] of the selected SIVsm genes; (2) express and accumulate SIV-Gv library; (3) verify incorporation of the SIVsm fragments into the surface borne C protein of Gv [gvpC]; and (4) evaluate functions of the members in terms of eliciting specific immune response and determining possible effects on cytokine production. Subsequently it will be possible to carry out initial testing of the particles in a murine system, rather than in primates, where the potential role of differentially displayed epitopes could be assessed in terms of generating protection against infection. Relevant findings can then be applied to epitope presentation and delivery in a sub-human primate model. Therefore, this system has the potential to provide knowledge about a novel approach, important to epitope specific vaccine development and to the utility of Gv as an antigen presentation and delivery system.

We present research aimed at determining how bacteria associated with aquatic plants influence phytoextraction of heavy metals. We focused on the aquatic plant Lemna minor and its response to cadmium (Cd) as a model. We studied bacterial communities from plants at a contaminated site, Rice City Pond (RCP), and plants provided by the U.S. Environmental Protection Agency (EPA) that had not been exposed to Cd. Initially, we performed comparative studies of the bacterial communities from these plants, with or without Cd addition, by analysis of 16S rRNA genes. We also studied Cd tolerance in both bacteria and plants in this system. Bacterial isolates were screened for metal tolerance and for the presence of Cd resistance genes, and we compared plant resposes to Cd between surface sterilized plants and plants with associated bacterial communities. In order to further determine the effects of bacteria on plants, sterile plants inoculated with bacterial isolates were grown in medium to enrich for bacterial growth. We measured plant growth rate, root length, final medium pH, bacterial biomass, and Cd concentrations in plants. We determined through 16S rRNA analyses that both EPA and RCP plants supported diverse bacterial communities. Upon Cd addition, EPA and RCP communities showed similar shifts in diversity, both becoming more diverse upon Cd addition. When investigating isolates from these treatments, we found that exposure to Cd increased culturability on Cd-amended medium; however, increased metal tolerance was not seen in all or even most isolates from treatments with Cd exposure, and Cd resistance systems were only found in a few isolates. We saw that surface-sterilized plants accumulated slightly higher concentrations of Cd than non-sterile plants. When enriched, bacteria slightly lowered the pH of the medium, but strong effects on root elongation and Cd phytoextraction. Bacteria seem to influence root elongation positively and influence Cd uptake negatively, keeping toxic concentrations of the metal from entering the plant. This study will allow better understanding of the role of aquatic plant-associated microbial communities in phytoremediation of heavy metals.

Identifying microbes possessing a synergistic combination of plant biomass breakdown and biofuel production are essential to develop effective strategies for producing next-generation liquid fuels. Clostridium phytofermentans is a novel, anaerobic soil microbe that produces ethanol as a primary product of fermentation during growth on plants such as switchgrass, corn stover and Brachypodium distachyon as well as on plant components such as cellulose, cellobiose, xylan, starch and glucose. A comprehensive understanding of its nutritional diversity, carbohydrate specificity along with identification of specific bottlenecks limiting growth and ethanol production is essential to the development of C. phytofermentans as a model biofuel organism. The work in this dissertation represents the first laboratory evolution experiment with a cellulolytic microorganism. For my dissertation research, I have used adaptive evolution as a tool to generate improved populations of C. phytofermentans with faster growth rate and ethanol producing capabilities when cultured on cellulose, cellobiose and xylan, as compared to the native strain. Whole-genome resequencing of the evolved populations was effective in identifying beneficial mutations in carbohydrate modules at varying levels of frequency. Analysis of mutations and protein modeling of mutated ABC transporter complexes detected mechanisms that may help to overcome constraints on carbohydrate metabolism in C. phytofermentans. These results reveal novel strategies for evolving and engineering cellulosic microorganisms for faster growth on plant biomass substrates. Based on the genome sequence of the native strain, known physiology and available experimental data, I also reconstructed the first genome-scale metabolic model for C. phytofermentans. Constraints on substrate uptake and ATP production were applied along with extensive manual curation and comparison with published clostridial models to bring model growth predictions closer to those observed under experimental conditions. The current model provides a systems-level view of C. phytofermentans metabolism and lays the groundwork for designing strategies to engineer a novel microbe to convert a broad range of biomass substrates to targeted biofuels of commercial interest.

Biogenic coalbed methane (CBM), an end product of anaerobic coal biodegradation, is a natural gas. It is generated and trapped in large quantities in deep, unmineable coalbeds. This dissertation describes a series of three integrated research studies to better understand the microbial players and trace element(s) limitations of methanogenic communities in this process. Microcosm experiments together with culture independent analyses demonstrated that hydrogenotrophic and methylotrophic methanogens are dominated by Euryarchaeota in CBM production water from our sampling site, Powder River Basin, Wyoming. Based on 16S rRNA gene clone libraries, Methanobacteria, Methanomicrobia , and Methanococci were major representatives of the methanogens. Betaproteobacteria and Gammaproteobacteria , Bacteroidetes, Firmicutes, and Actinobacteria were found as most abundant bacterial lineages. Trace elements are essential components of enzymes or cofactors of metabolic pathways associated with organic matter degradation and methanogenesis. CBM production water enrichments were amended with a mixture of eight essential trace elements (iron, nickel, cobalt, molybdenum, zinc, manganese, boron, and copper) at varying concentrations. Trace elements at optimized concentrations enhanced methane production by 37% relative to unamended controls. Furthermore, transcript levels of a molecular marker for methanogens, mcrA, correlated positively with elevated rates of methane production (R2=0.95). Additionally, trace element amendments to enrichments caused a shift in the metabolically-active methanogenic community. The addition of individual iron, nickel, zinc, manganese, tungstate, and selenium at their respective concentration gradients produced either no significant enhancement or had negative effects on methane production. However, addition of molybdenum (143.0 µg/L), cobalt (45.0 µg/L), and copper (15 µg/L) enhanced methane production by 72%, 55%, and 34%, respectively. The study revealed that molybdenum, cobalt, and copper were at limited concentrations in the CBM production water. Furthermore, addition of cobalt, molybdenum, and copper at their optimum concentrations for methane production caused an increase in methanogenic diversity and in transcript levels of mcrA (p<0.05). Knowledge of trace element limitations of the respective microbial community has a broader impact on our understanding of their ecology and physiology. Using coalbed methane, a fossil energy source that burns cleaner than coal or oil, would be an economically favorable alternative for the world's energy problems.

Soil and herbivore gut environments present different challenges to plant degrading bacteria in terms of nutrient availability, fluctuations in moisture, pH and temperature, and temporal constraints, however complex communities of microbes in each serve similar roles in hydrolyzing and fermenting the diverse components of plant biomass. This dissertation describes four projects with the underlying purpose to further understand the structure and functioning of anaerobic plant degrading communities. (1) A three-year microcosm experiment using enrichment and serial transfers to reduce the diversity of a complex soil community over time, tested the hypothesis that changes in community structure would be consistent across replicate samples and enabled the detection and isolation of persistent community members. (2) A second project to track the changes in bacterial community structure and function that occur during starch induced lactate acidosis in horses identified specific microbes that could be implicated in the recovery and/or resistance to these community changes. (3) Genomic data was used to compare clostridial species inhabiting the gut (belonging to the Lachnospiraceae, and Ruminococcaceae) and those that are free-living (belonging to the Clostridiaceae) to identify metabolic strategies that could enable specialization to a host associated or free-living lifestyle. (4) The genomic sequence analysis of Clostridium indolis, a member of the C. saccharolyticum species group, provided insights into the genetic potential of this poorly described taxa which will drive hypotheses regarding its metabolic and ecological activities and help to resolve distinctions between closely related taxa in this taxonomically confusing clade within the Lachnospiraceae.

Aerobic respiration generates unavoidable reactive oxygen species (ROS, detrimental by-products that damage biological molecules. The accumulation of ROS beyond the cellular capacity for detoxification results in oxidative stress. Bacteria have evolved transcription factors that sense ROS and transduce signals into differential gene expression. This study focuses on oxidative stress responses in Escherichia coli, an enteric bacterium and Pseudomonas aeruginosa, an important opportunistic human pathogen. E. coli activates the response to paraquat-induced superoxide stress through the SoxRS system. This study showed that transcription of pgi, a SoxRS regulated gene, and the corresponding phosphoglucose isomerase activity were induced in a SoxRS-dependent manner. A pgi null mutant was hypersensitive to paraquat when grown in rich medium with glucose. We provide evidence that pgi plays a role in maintaining NADPH for cells under oxidative stress by providing glucose-6-phosphate as a substrate for glucose-6-phosphate dehydrogenase. The opportunistic human pathogen P. aeruginosa has evolved under self-generated redox-cycling compounds, in addition to physiological and host-derived sources of oxidative stress. In an effort to identify novel redox sensor and effector proteins, we characterized the pqrCBAR genes. The mutation of the pqrCBAR genes resulted in hypersensitivity to oxidants, and pqrCBA form an operon under negative transcriptional regulation by PqrR. PqrR, a member of MarR family transcription regulators, has 4 conserved cysteines. Purified PqrR was found to exist as a dimer in solution, contain iron, and display characteristic UV spectra common to proteins with iron-sulfur clusters. DNAseI protection assay showed that PqrR bound in vitro at two distinct sites of the pqrA-pqrR intergenic region. PqrR binding activity was reduced by exposure to air, and reconstituted by DTT treatment. The site-directed mutagenesis of PqrR revealed that 3 conserved cysteine residues were essential for redox-sensing activity. To identify other novel antioxidant mechanisms in P. aeruginosa, we screened 15,003 of random transposon insertion mutants, and confirmed 15 mutants for paraquat sensitive phenotype.

Faithful DNA replication is essential for all organisms to maintain genetic integrity. During the DNA replication, replications forks are frequently stalled or collapsed due to the encounter of DNA lesions or blocking proteins. These events can occur anywhere on the chromosome which is away from the origin of replication. For survival, cells require a number of proteins to repair the damages and restart the replication near or at the damaged site. In Escherichia coli, a group of proteins called primosomal proteins consisting of PriA, PriB, PriC, Rep, DnaT, DnaC, DnaB and DnaG are required for directing DnaB replicative helicase back onto DNA substrates. The main difference between chromosomal replication at oriC and replication restart is the former process is initiated by DnaA recognizing a specific sequence of oriC while the latter process can be initiated by PriA recognizing a specific DNA structure. The mechanism of replication restart is highly ordered and well regulated, and to date, this mechanism has yet to be fully understood. This lab uses a genetic tool to understand replication restart in vivo and discovered that replication restart in E. coli can be explained by a multiple replication restart pathway model (Sandler, 2000). This dissertation presents work that advances our knowledge of replication restart by studying Rep and PriB using a genetic approach. Although it has been shown that Rep and PriB are important for replication restart, the contribution of these two proteins in vivo is still not clearly understood. In the case of Rep, this study provided the characterization of three rep mutants: a rep null mutant, a rep defective in a regulation of Rep monomer helicase activity and a rep ATP hydrolysis deficient mutant. Although these rep mutants showed a similar phenotype, there were some differences such as the phenotypes when combined with other mutations and spontaneous suppressors. In this study, a rep null mutant exhibited an unexpected phenotype including high basal levels of SOS expression and cell filamentation when combined with priB. The priB rep double mutant developed a spontaneous suppressor mapped in dnaC region. A rep mutant defective in an autoinhibition of Rep monomer helicase activity showed a more detrimental phenotype than a rep null mutant when combined with priB suggesting that this mutant Rep inappropriately removes PriC and completes with PriA. The other rep mutant, an ATPase deficient mutant, showed a similar phenotype to that of a rep null mutant as a single mutant and a double mutant with priB, however, spontaneous suppressors developed in the regions close to priC and dnaC, but not in the either of these genes. Lastly, PriB mutants that biochemical evidence has shown some effects on PriA, ssDNA and DnaT binding were tested for effects on replication restart in vivo. The studies of these PriB mutants lead to a model of the regulation of replication restart via PriA-PriB pathway which can be explained by a hand-off mechanism for primosomal assembly.