Browsing by Author "Robert Kelly, Committee Chair"

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    Biochemical and biophysical characterization of hyperthermophilic sugar isomerases and epimerases
    (2009-08-03) Harris, James Morgan; Robert Kelly, Committee Chair; Jason Haugh, Committee Member; David Ollis, Committee Member; Amy Grunden, Committee Member
    The availability of enzymes with optimal functional temperatures above 70°C has had considerable impact on industrial biocataysis. Extremely thermophilic enzymes have expanded the known thermal range of biological systems and ushered in a new era in applied-biocatalysis less restricted by limitations related to thermoactivity and thermostability. While advances have been in the made in understanding enzyme stabilization at high temperatures, much is to be learned of this complex biomolecular trait. Nonetheless, extremely thermophilic enzymes are being investigated as biocatalysts in a variety of bioprocesses. Here, the biochemical and biophysical properties of hyperthermophilicsugar isomerases and epimerases were examined with respect to their potential to mediate the biosynthesis of monosaccharides with nutritional and medical significance, also know as “rare sugars†. D-xylose-isomerase from Thermotoga neapolitana 5068 containing an N-terminal fusion with a chitin binding domain (ChiBD) from a hyperthermophilic-chitinase from Pyrococcus furiosus was examined incomparison to the wild type TNXI. The IM ChiBD TNXI half-life (19.9-h) was approximately three times longer than the soluble wild-type TNXI (6.8 h). Furthemore, the unbound soluble ChiBD TNXI had a longer life-life (56.5 h) than the immobilized enzyme. Both unbound and immobilized ChiBD TNXI not only had higher turnover numbers for glucose to fructose than the wild-type enzyme, but also for any known enzyme of this type. Molecular-modeling, based on structural information on the wild-type TNXI and PfChiBD, showed that the N-terminal fusion likely impacted subunit interactions, thereby contributing to the enhanced thermostability of the unbound ChiBD TNXI. These results illustrate that substantial changes in thermostability and reaction-kinetics can result from affinity tags for hyperthermophilic proteins. Sugar isomerases and epimerases (L-fucose isomerase (TMFI), L-arabinose isomerase TMAI), L-rhamnose isomerase (TMRI), D-tagatose-3-epimerase (TMTE), and D-xylose isomerase (TNXI)) from the hyperthermophilic bacterial genus Thermotogaawere examined as biocatalysts for rare sugar synthesis. Single and multi step reactions involving each isomerase with TMTE produced both expected and unexpected products, based on similar experiments with homologous mesophilic enzymes. The recently reported TMTE three-dimensional-structure revealed a non-conserved active site and hydrophobic binding pocket compared to mesophilic epimerases, likely responsible for the biocatalyic results observed in this study.
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    Carbohydrate Utilization Pathway Analysis in the Hyperthermophile Thermotoga maritima
    (2006-03-01) Conners, Shannon Burns; Todd Klaenhammer, Committee Member; Robert Kelly, Committee Chair; Greg Gibson, Committee Member; Bruce Weir, Committee Member; Jason Osborne, Committee Member
    Carbohydrate utilization and production pathways identified in Thermotoga species likely contribute to their ubiquity in hydrothermal environments. Many carbohydrate-active enzymes from Thermotoga maritima have been characterized biochemically; however, sugar uptake systems and regulatory mechanisms that control them have not been well defined. Transcriptional data from cDNA microarrays were examined using mixed effects statistical models to predict candidate sugar substrates for ABC (ATP-binding cassette) transporters in T. maritima. Genes encoding proteins previously annotated as oligopeptide/dipeptide ABC transporters responded transcriptionally to various carbohydrates. This finding was consistent with protein sequence comparisons that revealed closer relationships to archaeal sugar transporters than to bacterial peptide transporters. In many cases, glycosyl hydrolases, co-localized with these transporters, also responded to the same sugars. Putative transcriptional repressors of the LacI, XylR, and DeoR families were likely involved in regulating genomic units for beta-1,4-glucan, beta-1,3-glucan, beta-1,4-mannan, ribose, and rhamnose metabolism and transport. Carbohydrate utilization pathways in T. maritima may be related to ecological interactions within cell communities. Exopolysaccharide-based biofilms composed primarily of β-linked glucose, with small amounts of mannose and ribose, formed under certain conditions in both pure T. maritima cultures and mixed cultures of T. maritima and M. jannaschii. Further examination of transcriptional differences between biofilm-bound sessile cells and planktonic cells revealed differential expression of beta-glucan-specific degradation enzymes, even though maltose, an alpha-1,4 linked glucose disaccharide, was used as a growth substrate. Higher transcripts of genes encoding iron and sulfur compound transport, iron-sulfur cluster chaperones, and iron-sulfur cluster proteins suggest altered redox environments in biofilm cells. Further direct comparisons between cellobiose and maltose-grown cells suggested that transcription of cellobiose utilization genes is highly sensitive to the presence of cellobiose, or a cellobiose-maltose mixture. Increased transcripts of genes related to polysulfide reductases in cellobiose-grown cells and biofilm cells suggested that T. maritima cells in pure culture biofilms escaped hydrogen inhibition by preferentially reducing sulfur compounds, while cells in mixed culture biofilms form close associations with hydrogen-utilizing methanogens. In addition to probing issues related to the microbial physiology and ecology of T. maritima, this work illustrates the strategic use of DNA microarray-based transcriptional analysis for functional genomics studies.
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    Functional genomics analysis of carbohydrate conversion to biohydrogen by pure and mixed cultures of hyperthermophilic Thermotoga species.
    (2009-09-03) Gray, Steven Randall; Jason Haugh, Committee Member; Robert Kelly, Committee Chair; Amy Grunden, Committee Member; Balaji Rao, Committee Member
    The genus Thermotoga, is comprised fermentative anaerobes with optimal growth temperatures as high as 80°C. To understand the genetic and physiological diversity within this genus, the genome sequences of five Thermotoga species (T. maritima, T. neapolitana, T. sp. RQ2, T. petrophila, T. lettingae) were compared using bioinformatics tools. Except for T. lettingae, the genomes exhibited high degrees of homology and shared organizational traits. The in silico comparison was supported by genomic DNA cross-hybridization to a T. maritima cDNA microarray, where 83-94% of the probes in the three other Thermotoga species were recognized. These results indicated that the four Thermotoga species share a core genome (~1470 ORFs); ORFs unique to particular species likely reflect the influence of specific environmental or evolutionary factors. The significant homology among the four Thermotoga species facilitated development of a multi-species cDNA microarray for use in pure and mixed culture transcriptional response studies. The Thermotoga multi-species cDNA microarray was used to examine pure and mixed culture transcriptomes for growth on glucose and on a polysaccharide mixture. The multi-species array was used to estimate species composition of the mixed culture; composition varied from 6:1.5:1:1 for glucose batch culture to 2.3:2:2:1 in glucose continuous culture for T. sp. RQ2: T. maritima: T. petrophila: T. neapolitana, respectively. Composition in polysaccharide batch culture was similar to glucose continuous culture. Transcriptional response analysis provided clues to interspecies interactions. In glucose mixed culture, the ORFs encoding a phage tail-like bacteriocin (TM0785), lon proteases (TM1633, TM1869), E (TM1598), and a putative bacteriocin (TM1300) related to subtilosin A from Bacillus subtilis, were up-regulated relative to pure cultures. Differential regulation of several ORFs encoding HicAB Toxin-Antitoxin pairs (TM1310a-1313, TM1320-21) was noted, suggesting a potential role in interspecies interactions. Comparisons of growth on glucose and polysaccharides revealed changes in both core and non-core ORF transcription. All cultures exhibited upregulation of core genome xylan (TM0056-61, TM0070-77) and β-mannan utilization operons (TM1218-1223) on polysaccharide culture. An unclassified ABC transporter operon found only in T. neapolitana and T. sp RQ2 (TRQ2_0970-75) and a β-linked exopolysaccharide operon found only in T. maritima (TM0622-30) were up-regulated on glucose. A β-mannan utilization operon (TM1746-51), found only in T. maritima and T. sp RQ2, was up-regulated on polysaccharide culture. To investigate the potential of Thermotoga for biofuels production, biohydrogen generation through carbohydrate fermentation was examined for both pure and mixed Thermotoga cultures. T. maritima showed that, unlike the hyperthermophilic archaeon, Pyrococcus furiosus, which uses similar fermentative metabolism, sulfur had minimal effect on transcription. Furthermore, T. maritima preferred cellobiose over maltose, perhaps related to superior bioenergetics mediated by a cellobiose phosphorylase (TM1848) encoded in its genome. Volumetric H2 production rates (~1.2x10-3 mol H2 liter-1 hour-1) were similar for pure and mixed cultures, perhaps related to the function of metabolic pathways comprising the core genome. This work demonstrates the usefulness of multi-species arrays for examining closely related Thermotoga. The results indicted that, while differences in transcription were noted among pure and mixed cultures, culture growth and H2 production levels are not affected by species, substrate, or competition.
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    Transcriptional analysis of biofilm formation and stress response in hyperthermophilic microorganisms.
    (2004-03-24) Pysz, Marybeth Anne; David Olllis, Committee Member; Robert Kelly, Committee Chair; James Brown, Committee Member; Steven Peretti, Committee Member
    The significance of surface colonization and changing thermal conditions in hydrothermal environments motivated examination of biofilm formation and thermal stress response in the model heterotrophic hyperthermophilic microorganisms, Thermotoga maritima and Pyrococcus furiosus. Continuous culture, using maltose-based media and anaerobic conditions at 80°C for T. maritima and 95°C for P. furiosus, was used to generate dense biofilms on nylon mesh and polycarbonate filters; significant amounts of wall growth were observed in the chemostats for both organisms. Transcriptional analysis of biofilm- bound cells showed that genetic mechanisms observed for biofilm formation in less thermophilic bacteria applied to T. maritima. L-lactate dehydrogenase (TM1867), NADH oxidase (TM0379), sensor histidine kinase (TM0187), and TetR family transcriptional regulator (TM0823) were among the genes induced in T. maritima biofilms with mesophilic counterparts. Also consistent with cells in mesophilic biofilms was the differential expression of stress-related genes. Thermal stress genes, hrcA (TM0850), grpE (TM0851), and dnaK (TM0373) were up-regulated, indicating that elements of stress response are operational in hyperthermophilic biofilm environments. Expression of stress-related genes in the T. maritima biofilm prompted a study of stress response during heat shock at 90°C. A 407-gene targeted cDNA microarray was used to study the genetic differences between cells at 80°C and cells at 90°C after 0, 5, 10, 20, 30, 60, and 90 minutes. The two major heat shock operons dnaJ-grpE-hrcA (TM0849-TM0850-TM0851) and groEL-groES (TM0505-TM0506), as well as the genes encoding DnaK (TM0373) and heat shock protein class I (TM0374), exhibited maximal induction at early times (~5 minutes), subsequently decreasing to a steady-state level. This expression pattern has also been observed during heat shock of the mesophilic bacteria Escherichia coli and Bacillus subtilis. Also observed was the stress-related response of the SOS regulon involving usrB (TM1761) and recA (TM1859), and the down-regulation of this operon’s repressor lexA (TM1082). Atypical of heat shock response, the majority of genes encoding ATP-dependent proteases, including ClpP (TM0695), ClpQ (TM0521), ClpY (TM0522), LonA (TM1633), and LonB (TM1869), were down-regulated. ATPase Clp C subunits 1 (TM0198) and 2 (TM0873) were both up-regulated, along with ClpX (TM0146) and FtsH (TM0580). The ATP-independent heat shock serine protease HtrA (TM0571) was also induced. A number of other genes not related to stress response also showed significant changes in expression levels. These include transcriptional regulators, genes within the gluconate metabolic pathway, sugar transporters and glycosidases, and sigma factors. Homologs to E and A were induced during heat shock at 90°C, and suggesting that they are implicated in stress response regulation in T. maritima, although they have not been characterized to date. This work led to the development of chemostat-based methods for generating RNA from hyperthermophiles embedded in anaerobic biofilms that could be used for transcriptional analysis. Such analysis indicated possible connections between the genetic response of biofilm-bound cells and thermal stress response. The results here point to the significance of surface colonization and modification of cellular function arising from thermal changes in the microbial ecology of hydrothermal environments.