Browsing by Author "Amy M. Grunden, Committee Member"
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- Biochemical, Biophysical and Biotechnological Studies of Class II Xylose Isomerases from Hyperthermophilic Thermotoga Species(2005-10-15) Epting, Kevin Lee; David F. Ollis, Committee Member; Jason M. Haugh, Committee Member; Amy M. Grunden, Committee Member; Robert M. Kelly, Committee ChairXylose isomerase (XI) (D-xylose ketol isomerase, EC 5.3.1.5) is used to convert D-glucose to D-fructose in the production of high fructose corn syrup (HFCS). Here, the biochemical and biophysical properties of xylose isomerases from hyperthermophilic Thermotoga species are examined with regard to their potential for HFCS production at elevated temperatures. The effects of divalent metal cations on structural thermostability and inactivation kinetics of class II XIs from two mesophilic, one thermophilic, and one hyperthermophilic bacteria were examined. The three less thermophilic XIs were stabilized in the presence of Co²⁺ and Mn (and Mg²⁺ to a lesser extent), while the melting temperature of TNXI (T[subscript m]~100 degrees C) showed little significant variation. TNXI's kinetic inactivation was non-first order for all metal cases, and was modeled as a two-step sequential process. Unlike other class II enzymes examined, metals are required for TNXI activity but are not essential for structural thermostability. To determine if xylose isomerases from Thermotoga maritima (TMXI) and Thermotoga neapolitana (TNXI) could be utilized in HFCS production, the enzymes were compared with a commercial class I enzyme from Streptomyces murinus (SMXI) (Sweetzyme T™). While the soluble enzymes exhibited bi-phasic inactivation, the immobilized enzymes were characterized by a first order decay rate. A simple mathematical model was developed which utilizes the soluble enzyme kinetic data and immobilized inactivation rates to calculate productivities as a basis to compare enzymes under different process conditions. The extended N-terminus of class II XIs makes them attractive targets for attaching a carbohydrate-binding domain (CBD) for immobilization. Modifying the length of the N-terminal amino acid insert demonstrated that approximately half of the insert (to about residue 19) could be deleted while retaining activity; removing larger sections or the entire N-terminus caused the enzyme to misfold. A fusion protein (TNXI-CBD) with a thermostable CBD cloned from a hyperthermophilic chitinase (Pyrococcus furiosus 1233) attached to TNXI's N-terminus was created. The ability of the fusion protein to immobilize the enzyme to chitin beads was examined.
- Functional Genomic Analysis and Protein Expression in Lactobacillus(2008-12-12) Duong, Tri; Robert M. Kelly, Committee Member; Dahlia M. Nielsen, Committee Member; Amy M. Grunden, Committee Member; Todd R. Klaenhammer, Committee ChairThe lactic acid bacteria (LAB) are important in the production of food, industrial chemicals, and bulk ingredients. LAB, particularly probiotic lactobacilli, occupy important niches in the gastrointestinal tracts of humans and animals and are increasingly recognized as modulators of human and animal health. Major advances have been made in the genomic characterization of LAB facilitating their expanded use in bioprocessing and health. Trehalose is a cryoprotectant used to protect starter cultures from damage caused by freezing and lyophilization. Characterization of the tre locus of Lactobacillus acidophilus NCFM identified a trehalose PTS transporter, trehalose-6-phosphate hydrolase and a transcriptional regulator. Knockout mutants were used to determine that uptake and hydrolysis of trehalose is required for cryoprotection in L. acidophilus. Analysis of the FOS, lac and tre operons and pgm gene of L. acidophilus identified a number of putative promoter and repressor elements which were used to construct a series of expression vectors for use in lactobacilli. -glucuronidase reporter assays showed FOS, lac, and tre based vectors to be highly inducible by their specific carbohydrate and repressed by glucose. A construct based on the phosphoglycerate mutase (pgm) promoter was constitutively highly expressed. The development of these expression vectors is intended to support several novel applications including the delivery of vaccines and biotherapeutics by intestinal lactobacilli. The oxalate-degrading capabilities of lactobacilli have been studied with much interest in their potential use in a probiotic strategy for the management of hyperoxaluria and urinary stone disease. We describe the construction of a plasmid for the overexpression of the L. acidophilus NCFM oxalate degradation proteins, Frc and Oxc, and characterize its effect on oxalate degradation activity of L. acidophilus and Lactobacillus gasseri. This construct was able improve oxalate degradation by L. gasseri ATCC 33323 and complement an L. acidophilus Frc knockout mutant. Dendritic cells (DC) are antigen presenting cells found at mucosal surfaces that are important in directing acquired immunity. In this study, we construct an expression vector for recombinant DC-targeted Bacillus anthracis protective antigen for oral delivery by Lactobacillus acidophilus as a potential vaccine strategy against anthrax and evaluate its protective capability using a mouse model.
- Functional Genomic, Microbiological and Biochemical Characterization of Plant Biomass Deconstruction by the Extrememly Thermophilic Bacterium Caldicellulosiruptor saccharolyticus(2009-11-16) VanFossen, Amy; Jason M. Haugh, Committee Member; Robert M. Kelly, Committee Chair; David F. Ollis, Committee Member; Amy M. Grunden, Committee Member
- Functional Genomics Analysis of Biohydrogen Production by Hyperthermophilic Microorganisms(2008-05-09) Chou, Chungjung; Todd R. Klaenhammer, Committee Member; Jason M. Haugh, Committee Member; Robert M. Kelly, Committee Chair; Amy M. Grunden, Committee MemberThe tightening of fossil fuel supplies has generated interest in alternative energy sources in recent years. One primary focus is the conversion of biological feedstocks into biofuels, such as ethanol and hydrogen using anaerobic microorganisms. Efficient bioprocesses require insights into fermentative metabolism that can be facilitated by functional genomics. The arrival of the genomics era and advancement in system biology tools, such as DNA microarrays, has facilitated analysis of the transcriptomes of model microorganisms that could be used for bioenergy processes. In this work, the potential of using hyperthermophilic microorganisms, Pyrococcus furiosus and Thermotoga maritima, to produce biohydrogen and the underlying metabolic mechanisms that are used to accomplish this were investigated. Previous functional genomics efforts on global transcriptomics in P. furiosus and T. maritima focused on batch growth. However, the sensitivity of transcriptional response analysis makes it difficult to identify distinguish between key metabolic features and various secondary effects attributed to indirect impact on the growth status of the microorganism. To address this, an evaluation of the effects of growth phase, growth rate and cultivation method was undertaken for P. furiosus. Transcriptional data revealed excellent reproducibility between continuous cultures. Changes in growth phase in batch culture and dilution rate in continuous culture resulted in profound differences in aspects of cellular metabolism. Direct comparison between batch and continuous culture revealed differences between transcription of substrate utilization and stress response genes, such as heat shock proteins and anti-oxidative processes. Also examined were the effects of syntrophy between P. furiosus and the methanogenic hyperthermophile Methanococcus jannaschii growing in a chemostat setting. After evaluation of these basal transcriptome in P. furiosus, the effects of glucan linkage and the bioenergetics of elemental sulfur reduction were studied using continuous culture. The production rate of hydrogen and key fermentative products were measured and compared to the transcriptomes for various growth conditions. Interestingly, the utilization of different glucan substrates (α-linked maltose vs. β-linked cellobiose) not only affected the corresponding substrate transporters but also specific protein production, transcription of genes encoding membrane-bound hydrogenases, and trend toward H2S production in continuous culture. Bioenergetics parameters could be correlated to the transcriptional data which showed that the re-distribution of reductant flow was caused by glucan-regulated genes, such as alcohol dehydrogenases (PF0074-PF0075). Fianlly, continuous culture system was further utilized to study fermentative hydrogen production from xylose, glucose and xylose:glucose mixtures by hyperthermophilic bacterium T. maritima. Tryptone-supplemented xylose, glucose and xylose/glucose media were tested for hydrogen production in light of the corresponding global transcriptional profile. The results indicated that xylose-grown culture had higher protein production rate, while glucose grown culture tended to produce hydrogen. Surprisingly, the mix of both substrates increased the overall carbon intake and produces more hydrogen than what would be expected by linear extrapolation from data obtained in the pure substrate scenario. The transcriptional analysis revealed that the genes encoding enzymes in non-oxidative pentose phosphate pathway and the xylose transporter was the basis for this difference. The unexpected increase in the H2 production may be correlated to both the interaction between the pentose and hexose assimilation pathways and the efficiency of the carbohydrate-specific transporters.
- Functional genomics analysis of metal mobilization by the extremely thermoacidophilic archaeon Metallosphaera sedula(2010-04-20) Auernik, Kathryne Sherlock; Jason M. Haugh, Committee Member; David F. Ollis, Committee Member; Amy M. Grunden, Committee Member; Robert M. Kelly, Committee ChairAUERNIK, KATHRYNE SHERLOCK. Functional genomics analysis of metal mobilization by the extremely thermoacidophilic archaeon Metallosphaera sedula. (Under the direction of Dr. Robert Kelly.) Biomining processes recovering base, strategic and precious metals have predominantly utilized mesophilic bacteria, but relatively low yields have impacted wider application of this biotechnology. However, the use of high temperature microorganisms offers great potential to increase metal mobilization rates. Metallosphaera sedula (Mse) is an extremely thermoacidophilic archaeon with bioleaching capabilities, although little is known about the physiology of this microorganism. To better characterize Mse, its genome was sequenced and a whole genome oligonucleotide microarray was constructed for transcriptional response analysis. The physiological and bioenergetic complexities of Mse bioleaching were studied focusing on iron oxidation, sulfur oxidation, and growth modes (heterotrophy, autotrophy, and mixotrophy). The transcriptomes corresponding to each of these elements were examined for clues to the mechanisms by which Mse oxidizes inorganic energy sources (i.e. metal sulfides) and fixes CO2. Quinol/terminal oxidases important for maintaining intracellular pH and contributing to ATP generation via proton pumping were stimulated by different energy sources. The soxABCDD’L genome locus (Msed_0285-Msed_0291) was stimulated in the presence of reduced inorganic sulfur compounds (RISCs) and H2, while the soxNL-CbsABA cluster (Msed_0500-Msed_0504) was induced by Fe(II). Two similar copies of the SoxB/CoxI-like cytochrome oxidase subunit, foxAA’ (Msed_0484/Msed_0485) were implicated in fox cluster oxidation of Fe(II), as well as other energy sources. The doxBCE locus (Msed_2030-Msed2032) did not respond uniformly to either Fe(II) or RISCs, but was up-regulated in the presence of chalcopyrite (CuFeS2). A similar response was also observed for a putative rusticyanin (Msed_0966, rus), thiosulfate: quinone oxidoreductase (Msed_0363/Msed_0364, doxDA), and a putative sulfide:quinone oxidoreductase (Msed_1039, sqr), all three of which are candidates to serve as primary electron acceptors from inorganic substrates. Putative proteins implicated in the generation of reducing equivalents were identified (Dms/Sre-like reductase and Hdr-like reductases). Mixotrophy in Mse was defined as a strong preference for organic carbon combined with concomitant use of multiple inorganic (and organic) energy sources, if available. This growth mode was observed during CuFeS2 bioleaching, with organic carbon most likely obtained via recycling of lysed cell material.
- Hydrogen Metabolism in Campylobacter jejuni(2004-07-19) Borden, Nathan Joseph; Amy M. Grunden, Committee Member; Michael Hyman, Committee Member; Jonathon W. Olson, Committee ChairCampylobacter jejuni is an important human pathogen. It is the primary cause of acute gastroenteritis in the United States with an estimated 2-10 million cases annually. C. jejuni is a commensal colonizer of the avian intestines and contaminated poultry is the primary source of human infection. C. jejuni has a unique metabolism and respiratory chain which offer possible targets for antimicrobials. This research project explores the physiological role of hydrogenase in C. jejuni. C. jejuni contains an uptake hydrogenase that oxidizes H2 at approximately 40 nmols H2 oxidized/min/108 cells. H2 permits growth beyond C. jejuni's microaerophilic limits. In serum bottles containing 25% O2 and 10% H2 wild-type C. jejuni reaches a terminal OD600 of .695 while cells grown at 25% O2 are unable to grow and have a terminal OD600 of .0035. The ability to grow at high O2 atmospheres supplemented with H2 does not occur in a mutant strain deficient in hydrogenase activity. Hydrogenase could offer respiratory protection to O2-sensitive enzymes. Growth under H2 -supplemented atmospheres also enhances growth rate. Wild-type C. jejuni grown in batch culture without H2 had a generation time of 1.8 hours while cells grown in the presence of H2 had a generation time of .96 hours. Mutant C. jejuni, unable to oxidize H2, did not show such a drastic reduction in generation time. The ability of wild-type and mutant C. jejuni to colonize the avian intestine were compared and no significant differences resulted. Overall it appears H2 is an efficient supplemental source of energy that may provide a competitive advantage in the avian intestine.
- The Role of Ferric Uptake Regulator in Regulation of Metal Homeostasis, Metabolism, Virulence, and Protection Against Hydrogen Peroxide in Salmonella enterica serovar Typhimurium.(2010-01-19) Troxell, Stephen Bryan; Dr. Hosni M. Hassan, Committee Chair; Amy M. Grunden, Committee Member; Jon W. Olson, Committee Member; Paul E. Orndorff, Committee MemberOur goal was to determine the role of the major transcriptional regulator of iron metabolism in Salmonella enterica serotype Tyhphimurium. This regulator, termed Ferric Uptake Regulator or Fur, requires ferrous iron as a cofactor to bind DNA. Fur senses concentrations of intracellular ferrous iron and responds accordingly by binding the operator sites of promoters thereby inhibiting transcription of targeted genes, many encode proteins responsible for iron import. This method of regulation is lost when the cofactor is removed. Therefore, Fur controls iron concentration in the cell. In anaerobically grown Άfur, 298 genes were differentially expressed, 77 of which were previously identified as being under the control of a major regulator of anaerobiosis, Fumarate Nitrate Reduction or FNR. In general, increased transcription of genes required for iron acquisition/storage, carbohydrate metabolism, electron transport, oxidative/nitrosative stress, and modulators of virulence occurred in Άfur. Fur regulates transcription of a NO• detoxifying gene, hmpA, and a ferritin-like gene, ftnB, in an apparently indirect manner. In addition, Fur’s contribution to the regulation of Salmonella Pathogenicity Island 1 was discovered to be mediated by the derepression of a gene encoding a histone-like protein, hns, in Άfur. Bioinformatic analysis identified a putative Fur binding site upstream of hns and when the DNA segment containing this putative Fur site was deleted the result was significantly reduced expression of the H-NS repressed hilA in a fur+ background. This indicates increased expression of hns leading to reduced expression of hilA due to the inability to repress hns by Fur. Fur was found essential for virulence in an acute systemic model of Salmonella infection. Here we also report Fur is required for anaerobic activity of the two heme containing catalases (HPI and HPII) in Salmonella enterica serotype Typhimurium. Data presented indicate partial restoration of HPI and HPII in Άfur by adding δ-amino-levulinic acid, a precursor to heme biosynthesis, to the growth media. However, the addition of L-glutamate, a precursor to δ-amino-levulinic acid, was capable of partially restoring only HPI and had no effect on HPII activity. Likewise, supplying fur in trans partially restored HPI and HPII activity. In addition, Fur is required for maintaining anaerobic activity of HPI and HPII activity during H2O2 stress. Άfur exhibited reduced aerobic growth when grown in the presence of sodium cyanide, an inhibitor of heme containing respiratory enzymes. Therefore, Fur controls heme biosynthesis through modulation of L-glutamate and δ-amino-levulinic acid concentrations in the cell by protein abundance or enzyme activity of factors responsible for their synthesis. Data demonstrates that Fur controls a diverse set of genes and enzymes involved in physiology within the bacterial cell. This signifies the importance of metal homeostasis on the physiological state of the cell. However, the major method of regulation within the cell by Fur is indirect. In this regard, Fur acts as a sensor to controlling many factors which subsequently respond by modifying transcriptional expression, protein function, and/or enzyme activity. This demonstrates that Fur is a bona fide global regulator in Salmonella enterica serotype Typhimurium.
