Browsing by Author "Robert M. Kelly, Committee Chair"

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Biochemical and biophysical characterization of compartmentalizing proteases from the hyperthermophilic microorganism Pyrococcus furiosus
(2004-06-10) Chang, Lara Samofal; Steven W. Peretti, Committee Member; David F. Ollis, Committee Member; Todd R. Klaenhammer, Committee Member; Robert M. Kelly, Committee Chair
Proteases catalyze the cleavage of peptide bonds in peptides, polypeptides, and proteins using a hydrolysis reaction. From a biological standpoint, these enzymes are critical for cellular survival, particularly in removal of denatured proteins during stress events or of proteins that have completed their functions. Various proteases play distinct roles in the degradation of proteins, including proteinases that break down proteins and peptidases that break down the resulting oligopeptide products to single residues. The hyperthermophilic versions of proteases are useful for several reasons: they are easier to study because of their relative structural simplicity and, compared to their mesophilic counterparts, they are more stable in harsh conditions such as high heat. The focus of this study was on the biochemical and biophysical characteristics of two multi-subunit compartmentalizing proteases from the hyperthermophilic archaeon Pyrococcus furiosus (T[subscript opt]=100°C). The first protease was an oligopeptidase, PfpI (Pyrococcus furiosus protease I), and the second was a proteinase, called the proteasome. Both proteases are ubiquitous in all domains of life. However, they are theorized to have distinctly different roles within P. furiosus. The proteasome may be one of the primary proteinases, with access to its active sites tightly controlled by ATPase regulators that appear to be dependent on cellular environment. In contrast, the role of PfpI may be degradation of the smaller peptides that result from proteasome and other proteinase action. PfpI is a homo-multimer of 18.8-kDa subunits that assemble into hexameric rings. These rings then stack to form dodocamers and higher forms, with three active sites buried in hindered positions within each ring. Trimer, hexamer, and dodecamer forms were purified separately, with the dodecamer at least three-fold more specifically active than the smaller forms. It was also found that PfpI was only able to cleave oligopeptides up to 17 residues, preferring aromatic residues at the P₁ position. As the substrate length was increased, the cleavage by PfpI became less specific and confined to the C- and N-termini. The precise role of PfpI in P. furiosus still remains to be determined, with a particular need for studies of recombinantly expressed versions. The 20S proteasome, along with a theorized ATP-dependent regulator PAN (proteasome-activating nucleotidase), was investigated from several angles. Both enzymes, including native and recombinant forms, were tested for biochemical and biophysical characteristics as isolated structures and in combination. In particular, the PAN ATPase activity was tested primarily to observe its effects on different forms of the proteasome. Furthermore, both were subjected to targeted cDNA microarray experiments during heat shock of native P. furiosus. The P. furiosus proteasome was the first archaeal form investigated that contains two forms of the beta subunit instead of one. Subsequently, one of the primary focuses of the study was to elucidate the roles of the two (48% identical) beta subunits. Distinct differences in activity, stability, and level of ATPase-based stimulation were observed for the various proteasome forms. These differences were based on the presence or absence of one of the three subunits and the assembly temperature. The beta-2 subunit appeared to be the catalytic center for proteinase activity, while the beta-1 subunit played a stabilizing role. PAN was able to stimulate the native form of the proteasome during degradation of polypeptides but inhibited the native heat-shocked form in the same reactions. It was concluded that PAN, which is highly up-regulated during heat shock, may stimulate the native proteasome form, while the heat-shocked proteasome (containing higher levels of beta-1) may associate with a different set of regulating proteins.
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.
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.
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.
Physiological, Biochemical and Biotechnological Characterization of Glycoside Hydrolases from the Hyperthermophilic Bacterium
(2003-11-19) Chhabra, Swapnil R; Robert M. Kelly, Committee Chair; David F. Ollis, Committee Member; Denns T. Brown, Committee Member; Saad A. Khan, Committee Member
The genome sequence of Thermotoga maritima MSB8, encodes for the highest number of glycoside hydrolase genes amongst hyperthermophilic Bacterial and Archaeal genome sequences reported to date. The ability of T. maritima to utilize the polysaccharides galactomannan and CM cellulose as carbon sources can be attributed at least in part due to the presence of the genes cel5A (TM1751), man5 (TM1227) and cel74 (TM0305). The encoded proteins Tm Man5 and Tm Cel74 are extracellular marked by the presence of N-terminal signal peptides whereas Tm Cel5A is intracellular. Biochemical properties of recombinant versions of Tm Man5 and Tm Cel74, expressed in Escherichia coli, correlated well with predictions made by sequence comparisons. Thus, Tm Man5 was found to be a strict -mannanase while Tm Cel74 was found to be a strict endoglucanase. In contrast, although Tm Cel5A shows sequence similarity to an endoglucanase, its biochemical characteristics point to dual substrate specificity such that Tm Cel5A was found to hydrolyze both -mannan and -glucan polysaccharides. Glu-137 (proton donor) and Glu-253 (nucleophile) were found to be the catalytic residues in Tm Cel5A while Glu-329 was the catalytic nucleophile in Tm Man5. A mutation of these residues in each protein resulted in a complete loss of hydrolytic activity. Currently, Tm Cel74 is the only endoglucanase in Family 74 of glycoside hydrolases that lacks the presence of a cellulose-binding module at its C- terminus. Fusion of a binding module to the C-terminus of Tm Cel74 allowed the chimeric protein to bind and hydrolyze ii microcrystalline cellulose. Gene expression profiles of cel5A and man5 using Northern hybridizations and cDNA microarrays suggested co-regulation during growth on mannose and -1,4 mannan polysaccharides. Overall expression levels of cel74 were several fold lower than the other extracellular endoglucanase gene cel12A (TM1524) during growth on -1,4 glucan polysaccharides. Global gene expression analysis using a targeted cDNA microarray indicated the presence of tight regulatory mechanisms for glycoside hydrolase expression in T. maritima during growth on different carbon sources. Mixed model data analysis revealed co-regulation of genes within potential operons as well as sets of spatially distant gene strings with similar expression profiles, suggesting the presence of regulons in the T. maritima genome. This information in conjunction with biochemical characteristics of encoded proteins, was used to predict pathways for polysaccharide uptake and utilization in T. maritima. The research presented in this work provides a framework for future studies using full genome microarrays of T. maritima and other hyperthermophiles for the identification of glycoside hydrolases with novel sequences and substrate specificities.