Browsing by Author "Jason M. Haugh, 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 Chair
Xylose 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.
Computer Simulations of Protein Folding and Aggregation
(2004-05-13) Nguyen, Hung Duc; Carla Mattos, Committee Member; Steven W. Peretti, Committee Member; Jason M. Haugh, Committee Member; Carol K. Hall, Committee Chair
Computer simulation is used to study the competition between protein folding and aggregation, especially the formation of ordered structures that are also known as amyloid fibrils. Employing simplified protein models, we simulate multi-protein systems at a greater level of detail than has previously been possible, probe the fundamental physics that govern protein folding and aggregation, and explore the energetic and structural characteristics of amorphous and fibrillar protein aggregates. We first tackle the aggregation problem by using a low-resolution model called the lattice HP model developed by Lau and Dill. Dynamic Monte Carlo simulations are conducted on a system of simple, two-dimensional lattice protein molecules. We investigate how changing the rate of chemical or thermal renaturation affects the folding and aggregation behavior of the model protein molecule by simulating three renaturation methods: infinitely slow cooling, slow but finite cooling, and quenching. We find that the infinitely slow cooling method provides the highest refolding yields. We then study how the variation of protein concentration affects the refolding yield by simulating the pulse renaturation method, in which denatured proteins are slowly added to the refolding simulation box in a stepwise manner. We observe that the pulse renaturation method provides refolding yields that are substantially higher than those observed in the other three methods even at high packing fractions. We then investigate the folding of a polyalanine peptide with the sequence Ac-KA14K-NH2 using a novel off-lattice, intermediate-resolution protein model originally developed by Smith and Hall. The thermodynamics of a system containing a single Ac-KA14K-NH2 molecule is explored by employing the replica exchange simulation method to map out the conformational transitions as a function of temperature. We also explore the influence of solvent type on the folding process by varying the relative strength of the sidechain's hydrophobic interactions and backbone hydrogen bonding interactions. The peptide in our simulations tends to mimic a real polyalanine in that it can exist in three distinct structural states: alpha-helix, beta-structure, and random coil, depending upon the solvent conditions. We next examine the formation of fibrillar protein aggregates, which have been implicated in the pathology of several neurodegenerative diseases including Alzheimer's and Parkinson's, using the Smith/Hall intermediate-resolution protein model. Simulations were conducted on systems containing 12 to 96 Ac-KA14K-NH2 peptides at a wide variety of concentrations and temperatures. We are able to observe the formation of fibrils from random coils within just a few days on a single processor of an AMD Athlon MP 2200+ workstation. To our knowledge, these are the first simulations to span the whole process of fibril formation from the random coil state to the fibril state on such a large system. We find that fibril formation strongly depends upon the peptide concentration, the temperature, and the hydrophobic interaction strength of non-polar sidechains. The fibrils observed in our simulations mimic the structural characteristics observed in experiments in that most peptides in our fibrils were arranged in an in-register parallel orientation with intra-sheet and inter-sheet distances that are similar to those observed in experiments, and are disproportionately long along the fibril axis with about six beta-sheets, each of which contains many peptides. We also investigate the kinetics of fibril formation by performing constant-temperature simulations on systems containing 48 Ac-KA_14K-NH2 peptides with the Smith/Hall intermediate-resolution protein model. We find that fibril formation is nucleation dependent with an ordered nucleus of two beta-sheets, each with two to three peptides. The lag time before fibril formation commences decreases with increasing concentration and increases with increasing temperature. In addition, fibril formation appears to be a nucleated conformational conversion process in which small amorphous aggregates --> beta-sheets --> ordered nucleus --> subsequent rapid growth of a stable fibril. Fibril growth in our simulations involves both beta-sheet elongation, in which the fibril grows by adding individual peptides to the end of each beta-sheet and lateral addition, in which the fibril grows by adding already-formed beta-sheets to its side. Moreover, the rate of fibril formation increases with increasing concentration and decreases with increasing temperature. Finally, we examine the thermodynamics of systems containing 96 Ac-KA14K-NH2 peptides by performing replica exchange simulations over a wide range of temperatures and peptide concentrations. We map out a phase diagram in the temperature-concentration plane delineating the regions where random coils, alpha-helices, beta-sheets, fibrils, and amorphous aggregates are stable.
Fc-binding Hexamer Peptide Ligands for Immunoglobulin Purification
(2008-08-18) Yang, Haiou; Carla Mattos, Committee Member; Patrick V. Gurgel, Committee Member; Jason M. Haugh, Committee Member; Ruben G. Carbonell, Committee Chair; Peter K. Kilpatrick, Committee Member
Antibodies and their fragments have found a wide array of applications as pharmaceutical compounds, in addition to their common usage in the purification and localization of proteins. Antibody-based therapeutics accounts for roughly 20% of the therapeutic products in development in the USA, with the majority being of the immunoglobulin G (IgG) isotype. For IgG purification, affinity chromatography has been greatly used where Staphylococcus aureus Protein A and Streptococcus Protein G are the most common affinity ligands for IgG. However, the drawbacks associated with these two proteins have given rise to the searching for alternative affinity ligands for antibody purification. We therefore set our research goal as to find a small peptide that can bind hIgG through its Fc portion and can be used in purification of antibodies and Fc-fusion proteins. Small peptides are interesting for their advantages of being more stable, less immunogenic, less expensive and milder in elution than protein ligands. Peptides were searched by a radiolabeled-screening technique in a combinatorial linear hexamer peptide library built on solid phase Toyopearl AF-Amino resins. The screening identified a family of ligands with homogenous composition of His + aromatic group + positively charge group having the ability to match Protein A in binding human IgG (hIgG) through its Fc portion. The selectivity to the Fc portion is comparable to Protein A. The HWRGWV ligand of the Fc-binding peptide family has been investigated in many aspects and exhibits some interesting tributes. It has broad binding spectrum. It can bind all subclasses of hIgG, human IgD, IgE, IgM, and to a less extent human secretory IgA. It also displays the ability to retain chicken and several mammalian IgGs. Deglycosylation of hIgG has no influence on its binding to the HWRGWV ligand and the ligand does not compete with Protein A or Protein G in binding hIgG. It is suggested by the mass spectrometry data that HWRGWV binds to the pFc portion of hIgG and interacts with the amino acids SNGQPEN in the loop Ser383 — Asn389 by specific interactions. The selectivity of the HWRGWV resin to Fc over Fab is affected by its peptide density which also influences the equilibrium constant for hIgG. Increasing density increases both the association constant which is in the order of 105 M-1 and the binding of the Fab fragment. A ligand density of around 0.1 meq—g was determined to have both the specificity and appropriate affinity to Fc. HWRGWV demonstrates the ability to purify IgG. It can isolate hIgG from mammalian cell culture media containing 10% fetal calf serum (cMEM) with more than 95% of both purity and yield. The ligand was also used to isolate hIgG from Cohn II+III paste and an yield of 82% and purity of 73% were obtained in one step. The bound IgG can be recovered using phosphate buffer at pH 4 and its binding capacity for hIgG is 130 mg⁄g-dry-resin. Acetylation of the N-terminal amine does not pose any influence on either the static binding or the dynamic isolation of hIgG. Temperature has no significant influence on hIgG isolation from cMEM. Increasing peptide density improve the yield but with a compensation of the purity to a similar degree. Feed hIgG concentration in the range of 0.5 — 10 mg⁄mL affects the recovery yield where the yield is favored with higher IgG concentration. The separation of hIgG from cMEM by HWRGWV is comparable to Protein A at an initial hIgG concentration of 10 mg⁄mL and to A2P agarose gel at both 10 and 0.5 mg⁄mL, but using a milder pH 4 elution condition. HWRGWV was immobilized on Sepharose CL-4B. HWRGWV-modified Sepharose CL-4B can isolate hIgG from cMEM with similar purity to and a lower yield than on Toyopearl AF-Amino under the conditions optimized on the latter matrix. Our experiments also show that 2% AcOH is not strong enough to completely remove the bound proteins on HWRGWV. A better column regeneration and sanitation procedure is needed for longer column lifetime. This work demonstrates the possibility to use a peptide as short as six amino acids to mimic Protein A in IgG isolation by binding through the Fc portion.
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 Member
The 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 Chair
AUERNIK, 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 soxABCDDâ€™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, foxAAâ€™ (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.
Hybrid Particle-Nonwoven Membrane Materials for Bioseparations
(2009-07-07) Herigstad, Matthew Omon; Ruben G. Carbonell, Committee Chair; George W. Roberts, Committee Member; Jason M. Haugh, Committee Member; Behnam Pourdeyhimi, Committee Member; Patrick V. Gurgel, Committee Member
Adsorption separations performed in feed streams containing large particulates pose interesting problems, the solution of which would aid in many fields of bioseapartions. Production of biologically derived protein products is one of the most rapidly expanding sectors in the global economy. The capture and purification of these products has, of late, become the bottleneck of the industry and can account for approximately 50-80% of the production costs. The biopharmaceutical industry has begun to focus on improving overall economics by merging two or more separation schemes into one. The majority of the emphasis has been on combining the initial protein capture and host cell clearance steps; however, many of the currently available methods have shown little efficacy at large-scale. Additionally, interest in the clearance of pathogenic activity, most importantly infectious prions, from blood and blood derived products has grown over the past decade with the increased threat of blood-transfusion of variant Creutzfeldt-Jakob disease. This work characterizes the transport and binding properties of a novel hybrid particle-nonwoven membrane medium in which a polymeric chromatographic resin is entrapped between layers of a nonwoven polypropylene membrane (a particle-impregnated membrane or PIM). This membrane-supported resin construct offers the advantage of increased interstitial pore diameter to allow passage of cells and other debris in the feed, while providing sufficiently high surface area for product capture within the resin particles. Columns packed with stacked disks of PIM displayed excellent flow distribution, and had an interstitial porosity of ÃŽÂµb = 0.48 Ã‚Â± 0.01, a 25-60% increase over those typically observed in a packed bed. These columns were able to pass over 95% of E. coli cells and human red blood cell concentrate (RBCC) in 30 column volumes, while maintaining a pressure drop significantly lower than that of a packed bed. The dynamic binding capacity of the chromatographic resin entrapped in the PIM packed column for bovine serum albumin (BSA) was essentially the same as that observed with the same volume of resin in a packed bed. Additionally, the binding of prion was characterized to PIM constructs containing an affinity ligand for PrPSc, in saline, RBCC, and human IgG solutions. The General Rate (GR) model of chromatography was used to analyze experiments indicating that the breakthrough and elution behaviors of the PIM column are predictable, and very similar to those of a normal packed bed. These results indicate that PIM constructs can be designed to process viscous mobile phases containing particulates while retaining the desirable binding characteristics of the embedded chromatographic resin. The PIM systems could find uses in adsorption separation processes from complex feed streams such as whole blood, cell culture, and food processing and could offer a process alternative to expanded beds.
Identification, Characterization, and Physiologic Analysis of Proteolytic Enzymes in Hyperthermophilic Organisms
(2008-12-06) Michel, Joshua Klaus; Robert M. Kelly, Committee Chair; Todd R. Klaenhammer, Committee Member; David F. Ollis, Committee Member; Jason M. Haugh, Committee Member
Capable of growth at or above 80°C, hyperthermophilic organisms encode a myriad of proteolytic enzymes, including a number of homo- and hetero-multimeric complexes. These large hyperthermophilic proteases are often comprised of fewer distinct subunits compared to the less thermophilic bacterial and archaeal homologs; thus they provide an attractive model system for study. Whole genome transcriptional response analysis was used to survey both previously characterized and putative proteases in the hyperthermophilic archaea Pyrococcus furiosus and Sulfolobus solfataricus and hyperthermophilic bacterium Thermotoga maritima. The proteolytic transcriptional response of these three organisms demonstrated a complex synergistic relationship between the ATP-dependent proteases (responsible for initial degradation of proteins) and the ATP-independent proteases that liberate free amino acids from smaller peptides. Additionally, all three proteolytic systems showed up-regulation of protease genes involved in the degradation of misfolded and regulatory proteins during cellular stress response to changes in environmental pH and temperature. To a lesser extent, the ATP-dependent proteases (e.g. Clp) were also involved in the response of T. maritima to increased levels of extracellular acetate; this was accompanied by decreased transcription of metabolic genes and entry into stationary-phase. Thermal stress conditions also affected expression and multi-subunit composition in the P. furiosus proteosome, yielding a more thermostable complex. The P. furiosus genome encodes three proteasome component proteins: one α (PF1571) and two β proteins (β1-PF1404; β2-PF0159), as well as an ATPase (PF0115), referred to as Proteasome-Activating Nucleosidase (PAN). Proteosome assembly and characteristics were found to be highly dependent on the environmental growth conditions. Increased growth temperature (shift from 90 to 105°C) resulted in a 2-fold up-regulation of β1 mRNA within five minutes, suggesting a specific role during thermal stress. Consistent with this data, two-dimensional SDS PAGE revealed that incorporation of the β1 protein relative to β2 into the 20S proteasome (or core particle, CP) increased with increasing temperature for both native and recombinant versions. The recombinant form of PFα+PFβ1+PFβ2 CP assembled at 105°C was found to be more thermostable and have different catalytic rates and substrate specificities, when compared with a recombinant form of PFα+PFβ1+PFβ2 assembled at 90°C or the PFα+PFβ2 version assembled at either 90°C or 105°C. These results indicate that the β1 subunit in the P. furiosus 20S proteasome plays a thermostabilizing role in archaeal proteasome function during thermal stress when polypeptide turnover is essential to cell survival. In contrast to P. furiosus, the hyperthermophilic archaeon Archaeoglobus fulgidus produces a 20S proteasome comprised of two distinct subunits, α (AF0490) and β (AF0481). Combination of A. fulgidus &alpha and P. furiosus β1 and/or β2 yielded hybrid proteasome CPs that display characteristics different then the wild-type enzymes. Notably, A. fulgidus α was found to preferentially assemble with P. furiosus β1, even in the presence of AFα. The A. fulgidus recombinant proteasome exhibited comparable biochemical properties to the P. furiosus complex (α+β2 or &alpha+β1+β2), albeit with a reduced optimal temperature. However, the recombinant A. fulgidus 20S proteasome and hybrid CPs were not substrate-inhibited as was the case for the recombinant P. furiosus 20S proteasome. Taken together, these results demonstrate that proteasomes can be constructed with subunits from different hyperthermophiles, and that subunit composition influences biochemical and biophysical properties. The fact that hybrid inter-generic versions can be created in vitro also suggests that CPs in particular archaea may have arisen from common sources. Furthermore, the ability to interchange subunits and alter composition of the proteasome suggests that this system may provide a useful platform for designing proteases with unique activities or specific biophysical properties required for any biotechnological application.
Intercellular Communication in Hyperthermophilic Microorganisms
(2006-11-28) Johnson, Matthew Robert; David F. Ollis, Committee Member; Jason M. Haugh, Committee Member; James W. Brown, Committee Member; Robert M. Kelly, Committee Chair
In the microbial world it is becoming apparent that many syntrophic, symbiotic and competitive interactions that occur within and between species are driven largely by a form of cell-to-cell communication known as quorum sensing, in which communication within and between species occurs through the use of highly specific signal molecules. In this work, evidence is presented through functional-genomics approaches that cell-to-cell signaling is a phenomenon not limited to mesophilic bacteria. In Thermotoga maritima, a hyperthermophilic bacterium, a previously uncharacterized small peptide (TM0504) that is very highly expressed under syntrophic growth conditions was found to have quorum sensing properties, inducing the cell-density dependent expression of glycosyltransferases to form exopolysaccharides. Exopolysaccharide production enabled the close association of T. maritima to the hyperthermophilic methanogen Methanococcus jannaschii, underlying the synthrophic transfer of hydrogen between species. Upon further examination, it was found that distinct life cycles exist within this syntrophic relationship, with rapid growth and aggregation in the co-culture followed by detachment of the two species in stationary phase. This process is postulated to be driven by an unknown quorum sensing system, allowing the detachment and spread of these organisms into new growth environments. In addition, evidence was provided that showed that Pyrococcus furiosus, a hyperthermophilic archaeon growing optimally near 100°C, both produces and responds to a recognizable form of AI-2, a furanosyl borate diester and known universal autoinducer of quorum sensing in mesophilic bacteria. As P. furiosus and all other members of the Archaea lack the LuxS enzyme involved in AI-2 biosynthesis in mesophilic bacteria, an alternative pathway must be involved. Purification of native AI-2 biosynthetic enzymes from P. furiosus crude cell extracts using a biological reporter assay allowed for the isolation of fractionated cell-free extracts that could convert adenosine to a species that triggered quorum sensing in a reporter strain of Vibrio harveyi. Through the use of the available genome sequence, it was proposed the production pathway for AI-2 involves the phosphorylation of ribose from adenosine through the activity of a eukaryotic-like MTA-phosphorylase (PF0016). In fact, the recombinantly produced MTA phosphorylase could complement fractionated P. furiosus biomass to produce enhanced levels of AI-2 activity from adenosine at 90°C. Other components of the pathway are under investigation, but likely includes a ribose phosphoisomerase (PF1258) to produce phosphorylated ribulose, which can be non-enzymatically converted to (4S)-4,5-dihydroxy-2,3-pentanedione (DPD). A potentially unique contribution of thermal energy in the conversion is proposed as this step is significantly accelerated at hyperthermophilic temperatures over rates observed at mesophilic temperatures, suggesting temperature may have had a role in directing the evolution of cell-to-cell signaling systems. Overall, these results suggest quorum sensing phenomena occurs in hyperthermophilic microorganisms, where it likely plays an important role in regulating intra- and inter-species interactions and defining microbial phenotypes.
Stochastic Modeling of Transcription Factor Binding Fluctuations
(2004-08-22) Pirone, Jason R; Timothy C. Elston, Committee Co-Chair; Alun L. Lloyd, Committee Member; Charles E. Smith, Committee Co-Chair; Robert C. Smart, Committee Co-Chair; Jason M. Haugh, Committee Member
Cell populations typically exhibit binary or graded transcriptional responses to external stimuli. Transcription factor interactions with DNA have been hypothesized to account for both of these scenarios. To address this hypothesis, two stochastic models were constructed to describe transcription in simple, engineered eukaryotic systems. In the first system, three transcription factors bind independently to enhancer sites directing production of protein. This system has no regulation in the form of feedback loops, but the system nonetheless exhibits a clear binary response when transcription factor binding fluctuations are slow. The graded response occurs when transcription factor binding fluctuations are rapid. Thus, transcription factor binding fluctuation is an important mechanism underlying and reconciling the graded and binary transcriptional responses. In the second model, the influence of autoregulatory feedback loops on transcription was assessed. Autoregulated systems are capable of exhibiting bistability, a mechanism cited to explain the binary transcriptional response. In this autoregulated system, a dimeric protein acts as a transcription factor to increase its own production. Using biologically realistic parameter values, the system was determined not to be bistable. However, binary transcriptional responses were still observed in stochastic models due to discrete fluctuations in transcription factor binding. The results of both models suggest that transcription factor binding fluctuations play an important, and often overlooked role, in observed patterns of transcriptional activation.