Enantiopure alcohols are valuable intermediates in fine organic synthesis, in particular for preparation of biologically active compounds. The necessity of preparing single enantiomer drugs in an optically pure form has triggered much research, especially in the pharmaceutical industry. The biocatalytical production of chiral alcohols by alcohol dehydrogenase enzymes is characterized by the asymmetric reduction of the corresponding ketones, usually with high degree of stereoselectivity. The commercial value of the enzymes as stereoselective biocatalysts has been a significant driving force in understanding features that control their mechanism of catalysis and stereoselectivity. This work focuses on two enantiocomplementary dehydrogenase enzymes ((R)- and 2-(S)-hydroxypropyl-CoM (HPC) dehydrogenases (DH)) of the epoxide carboxylation pathway in Xanthobacter autotrophicus strain Py2. The main goal of this dissertation is to kinetically, mechanistically and structurally characterize S-HPCDH and through the comparison studies with R-HPCDH reveal the basis for high degree of stereoselectivity exhibited by both enzymes. Analysis of the molecular structure of R-HPCDH and the homology model of S-HPCDH suggests a mechanism of substrate specificity in which the binding of the substrate sulfonate moiety at distinct sites on each stereoselective enzyme directs the orientation of the appropriate substrate enantiomer for the hydride abstraction. The positively charged residues responsible for binding the CoM moiety of the substrate were identified in R-HPCDH (Arg152 and Arg196), and in S-HPCDH (Arg211 and Lys214). Site-directed mutagenesis confirmed their importance in binding and orienting physiological substrates, but not the substrates lacking the CoM moiety. Extensive kinetic and mechanistic characterization of S-HPCDH reveals its key catalytic features similar to those of R-HPCDH, but also points out a few important differences. Furthermore, the role of the methionine residues flanking the substrate in the active site of both dehydrogenases was investigated. Substitution of these residues to alanine resulted in enzymes with significantly altered catalytic parameters and suggested their importance in binding and catalysis. Additionally, the X-ray crystal structures of the Met187Ala and Met192Ala mutants of R-HPCDH have revealed their role as "gate keepers," protecting the active site from the surrounding solvent. Kinetic analysis of Met187Leu and Met192Leu mutants implied a structural, rather than catalytic function of the methionines. It is proposed that steric clashes of the terminal methyl group of the HPC substrates with the nicotinamide ring of NAD+ are a major determinant of the enantioselectivity in S-HPCDH. This research provides the first side-by-side characterization of a pair of short-chain dehydrogenase/reductase (SDR) enzymes expressed simultaneously to act on two enantiomers of the same alcohol produced in a metabolic pathway. The R-HPCDH and S-HPCDH enzymes are distinguished from all other known members of the SDR family in using the novel sulfonate functional group of coenzyme M as a handle for chiral discrimination. These results provide a standard for examining the molecular basis of stereoselectivity in other such enzyme pairs.

This thesis presents studies that examine microbial extracellular electron transfer that an emphasis characterizing how environmental conditions influence electron flux between microbes and a solid-phase electron donor or acceptor. I used bioelectrochemical systems (BESs), fluorescence and electron microscopy, chemical measurements, 16S rRNA analysis, and qRT-PCR to study these relationships among chemical, physical and biological parameters and processes. / Engineering and Applied Sciences

This work was undertaken with the objective of isolating and characterising the bdh gene of P. aeruginosa PAOI. Isolation of the bdh gene was initially attempted by PCR amplification and then by heterologous complementation of E. coli (LS5218) and S. meliloti (Rm11107) strains unable to catabolise 3-hydroxybutyrate. Three classes of plasmids were isolated. Class I comprised two plasmids, p5218-02 and p5218-07, isolated via complementation of LS5218, which were capable of complementing both LS5218 and Rm11107 for growth on 3-hydroxybutyrate. 3-hydroxybutyrate dehydrogenase (BDH) activity was not detected in an extract of LS5218 (p5218-02). The sole 3.6-kb EcoRI fist was partially sequenced and found to have three putative open reading frames (ORF). ORF 1 is homologous to the fusE gene of E. coli. We hypothesised that p5218-02 encodes an enzyme capable of degrading 3 hydroxybutyrate, but does not encode the bdh gene. Plasmids of class II (p30065) and class III (p30066) were isolated via complementation of Rm11107. Significant BDH activity was detected in an extract of Rm11107 (p30066), but not in Rm11107, leading to the hypothesis that p30066 carries the bdh gene.

Characterizing dissolved organic matter (DOM) composition remains a major unresolved problem in aquatic ecology. "Tryptophan-like" dissolved organic matter fluorescence (FDOM) was found to be a much better predictor of heterotrophic bacterial metabolism in 28 Quebec lakes than dissolved organic carbon (DOC), describing 52, 44, 51 and 55% of the variability in bacterial production (BP), bacterioplankton respiration (BR), total bacterial carbon consumption (TBCC), and total plankton community respiration (CR), respectively. In addition, the study provides indirect support for the view that FDOM represents a product of bacterial activity, rather than a bioavailable substrate. This is the first field study to show that fluorescence spectroscopy can be used to characterize an aspect of DOM composition that is related to bacterial metabolism, and provides results that encourage further exploration of the potential uses of DOM fluorescence spectroscopy as a predictive tool.

Microorganisms are increasingly used in toxicological studies to determine potential environmental impacts of xenobiotic compounds. A multi-stage laboratory model was developed to facilitate the examination of environmental impacts of selected pollutants on fundamental cycling processes inherent to aquatic ecosystems, namely, the degradation of organic substances and nitrogen transformations under aerobic conditions. A microbial association representative of riverine ecosystems was enriched for, isolated and cultured within the model. Characterisation of the microbial association were undertaken. Scanning electron microscopy and bright field microscopy revealed that a diverse heterogenous community of microorganisms had established within the model. Successional metabolic events, namely organic carbon catabolism, ammonification of organic nitrogen and the process of nitrification were differentiated in time and space with the microbial association integrity still being retained. The establishment of a microbial association within the model was primarily dependent on: dilution rates, specific growth rates and interactions between microorganisms and the prevailing environmental conditions. Growth-rate independent populations of microorganisms established within the model and were thought to contribute significantly to the metabolic processes within the model. Nitrifying activity was identified as a rate-limiting process within the model. Following separation of metabolic events, the ecotoxicological impacts of phenol and 2,4-dichlorophenol on the association were assessed. The biological oxidation of ammonia through to nitrate (nitrification) was found to be a sensitive indicator of perturbation. The model was found to be suitable for testing both acute and chronic intoxication by pollutant compounds as well as for biodegradation testing and the possible evaluation of ecotoxicological impacts of wastewater treatment plants. The main disadvantages of the model arose from its operational complexity, its empirical nature and its impracticality for screening large numbers of compounds. A bioassay based on the inhibition of ammonium oxidation was developed in order to fulfil the requirements for a simple and rapid test protocol for the initial screening of perturbant compounds. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1996.

Ovine (strains 9L and 3384Y) and bovine (strains 649, 2336 and 8025) isolates of Histophilus somni were investigated for their ability to acquire iron from haemoglobin (Hb). Bovine isolates were capable of utilizing bovine, but not ovine, porcine or human Hb as a source of iron. Ovine isolates could not obtain iron from Hb. Bovine isolates bound bovine, ovine, and human Hbs by means of the same iron-repressible receptor(s) and produced a ~120-kDa iron-repressible, outer membrane protein. Using PCR approaches, an iron-regulated operon containing hugX and hugZ homologues and a gene (hgbA) that encodes a TonB-dependent, Hb-binding proteins were identified in strains 649, 9L and 3384Y. In strains 9L and 3384Y, HgbA is truncated offering a possible explanation for their lack of utilization of Hb as an iron source. In strains 2336 and 8025, expression of HgbA was also subject to a form of phase variation.

The pathogen Vibrio cholerae uses cations as a primary currency of virulence and environmental persistence, using gradients of those cations to move, acquire nutrients, and control virulence gene expression. An understanding of the overlapping roles of bioenergetics and chemotaxis in the virulence and environmental survival of V. cholerae issues from a large body of prior work, but the interplay of each component is not yet clearly understood. To this end, the activity of the antiporters Vc-NhaP1, Vc-NhaA, and Vc-NhaB was assayed, as was the sodium transporting respiratory pump NQR, and environmental stimuli were paired with potential motilitylinked sensors. The Vc-NhaP1 antiporter was found to be a K⁺(Na⁺)/H⁺ antiporter essential for V. cholerae growth at low environmental pH. Deletion of the V. cholerae nhaP1 gene caused growth inhibition when external potassium was either limited (100 mM and below) or in excess (400 mM and above). This growth defect was most apparent at mid-logarithmic phase, after 4-6 hours of culturing. Using a pH-sensitive GFP protein, cytosolic pH was shown to be dependent on K⁺ in acidic external conditions in a Vc-NhaP1-dependent manner. When functionally expressed in an antiporterless E. coli strain and assayed in everted membrane vesicles, Vc-NhaP1 operated as an electroneutral alkali cation/proton antiporter, exchanging K⁺ or Na⁺ ions for protons within a broad pH range (7.25 to 9.0). These data establish the putative V. cholerae NhaP1 protein as a functional K⁺(Na⁺)/H⁺ antiporter of the CPA- 1 family that is required for bacterial pH homeostasis and growth in an acidic environment. Further, a model system comprised of a V. cholerae strain lacking both the nqr operon and the ORFs of Vc-nhaA or Vc-nhaB was generated and tested with and without lactate. These strains, along with the single mutants of nqr, Vc-nhaA, and Vc-nhaB, were assessed for aerobic growth as a function of media pH and cation concentration (Na⁺, Li⁺, or K⁺). Loss of Vc-NhaA and, to a lesser extent, Vc-NhaB, was better observed when NQR was absent but lactate was added to facilitate replenishment of the quinone pool. Loss of Vc-NhaA in this background inhibited growth most at basic pH under increasing Na⁺ and Li⁺ conditions, and loss of Vc- NhaB in this background inhibited was most severe in acidic conditions in the presence of 0-100 mM Na⁺ or Li⁺. We also observed the growth inhibition of Vc- NhaA in the absence of NQR and in the presence of lactate and 100-450 mM Li⁺, which has not been previously reported. These growth defects were restored upon expression of the cognate antiporter gene on an inducible expression vector. Lastly, potential chemotaxis stimuli were correlated with cognate methyl-accepting chemotaxis protein (MCP) receptors. The homology of MCP sensory domains among Vibrionaceae demonstrated a subset were unique to V. cholerae. Of these unique MCPs, transposon insertion in VC0098 significantly reduced chemotaxis swarm diameter towards Na⁺ and K⁺. Additionally, the MCP VCA0663 was shown, by transposon mutagenesis and complementation, to direct chemotaxis towards N-acetylglucosamine. Additional observations are described concerning the chemotaxis defects incurred by transposon mutagenesis of MCPs in vitro towards mucin, bile, or L-serine. MCP strains were also tested in vivo for 4 and 24 hours in the infant mouse model of infection. None of the observed chemotaxis defects showed complete loss of chemotaxis by transposon mutagenesis, in line with the hypothesis that the large number of MCPs encoded by V. cholerae result in redundant chemotaxis sensory functions. These findings add to the understanding of how bioenergetics and chemotaxis interact within V. cholerae, a foundation from which the bacterium can be understood and, eventually, controlled. / Graduation date: 2012

Thesis (doctoral)--Universiteit van Amsterdam, 1991. / Summary in Dutch. Includes bibliographical references (p. 94-102).

Made available in DSpace on 2015-04-11T13:55:41Z (GMT). No. of bitstreams: 1 Cassiane Minelli.pdf: 1637307 bytes, checksum: b5227c9445b1a0b3f8dcc8fcef9bcfd3 (MD5) Previous issue date: 2013-06-24 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / One of the biggest successes of Brazilian agriculture is the Pro-Alcohol, whose base of support is the culture of sugar cane, which leaves a lapse of activity in the offseason. In view of this, several alchool industries have been investing in other cultures, where the starch is the primary source for the alcohol production. The pursuit of amylase in thermotolerant Bacillus sp. And rhizobia from Amazonian soils is a strategy to be used for the bio-industries to reduce or stop importing them. Mutation was made from two Bacillus sp. containing amylases by exposure to ultraviolet light. Also tested were 40 strains of rhizobia for the presence of thermophilic amylases. Enzyme assays were performed to check for amylase activity at different temperatures, times, pHs, thermal shock and thermal stability. The Bacillus sp. ITAAM 023M mutant showed higher amylolyse than mutant ITAAM 010M. The amylases of the two Bacillus sp mutants and rhizobias proved quite diverse as their characteristics, indicating they are different from each other. The temperature with higher amylase activity of the mutants ITAAM 010M and ITAAM 023M was 100 ˚C. Amylase from the mutant ITAAM 010M showed greater activity in pH 4.5, and from the ITAAM 023M at pH 7.5. Their amylases showed high sensitivity to thermal shock. Amylase from mutant ITAAM 010M showed high thermostability at temperature of 90 ˚C. The amylase from ITAAM 023M, at a temperature of 80˚C. The highest activity shown by ITAAM 010M amylase was 1.6 U / ml and the ITAAM 023M, 1.5 U / mL. From the 40 rhizobia, 17 grew well in culture medium containing starch, 19 produced amylolytic halo, and 11 proved to be thermophiles. Rhizobia INPA INPA R001 and INPA R020 showed the highest rates of amylolyse. Their amylase showed greater activity at 90˚C. Amylase of INPA R001 showed higher activity at 100˚C, and from INPA R020, 30˚C. Amylase from INPA R001 showed greater activity at pH 4.5 and from INPA R020, at pH 7.5. Their amylases showed high sensitivity to thermal shock. Amylase from INPA R020 showed higher thermostability at 90 °C and from INPA R001 at 100˚C. The higher activity shown by amylase of rhizobia INPA R001 was 1.6 U/mL, and from INPA R020, 7.5 U/mL / Um dos maiores sucessos da agricultura brasileira é o Pró-Álcool, cuja base de sustentação é a cultura da cana-de-açúcar, que deixa um lapso de atividade na entressafra. Em vista disso, diversas usinas vêm investindo em outras culturas, onde o amido é a fonte primária para a produção do álcool. A busca de amilases termotolerantes em Bacillus sp. e em rizóbios de solos da Amazônia é uma estratégia a ser usada para que as bioindústrias reduzam ou parem de importá-las. Foi feita a mutação de dois Bacillus sp. contendo amilases com exposição à luz ultravioleta. Foram testadas também, 40 estirpes de rizóbio quanto à presença de amilases termotolerantes. Foram feitos ensaios enzimáticos para a verificação de atividade amilolítica em diferentes temperaturas, tempos, pHs, choque térmico e termoestabilidade. O mutante de Bacillus sp. ITAAM 023M mostrou maior índice de amilolise do que o ITAAM 010M. As amilases dos dois mutantes de Bacillus sp e dos rizóbios mostraram-se bem diversificadas quanto às suas características, indicando serem diferentes umas das outras. A temperatura com maior atividade das amilases dos mutantes ITAAM 010M e ITAAM 023M foi 100˚C. A amilase do mutante ITAAM 010M mostrou maior atividade em 4,5, e a do ITAAM 023M em pH 7,5. Suas amilases mostraram alta sensibilidade ao choque térmico. A amilase do mutante ITAAM 010M mostrou maior termoestabilidade na temperatura de 90˚C. A do ITAAM 023M, na temperatura de 80˚C. A maior atividade mostrada pela amilase do ITAAM 010M foi de 1,6 U/mL e a do ITAAM 023M, 1,5 U/mL. Dos 40 rizóbios, 17 cresceram bem em meio de cultura contendo amido, 19 produziram halo amilolítico e 11 se mostraram termofílicos. Os rizóbios INPA R001 e INPA R020 mostraram os maiores índices de amilolise. Suas amilases mostraram maior atividade a 90˚C. A amilase do INPA R001 mostrou maior atividade a 100˚C, e a do INPA R020 a 30˚C. A amilase do INPA R001 mostrou maior atividade no pH 4,5 e a do INPA R020 no pH 7,5. Suas amilases mostraram alta sensibilidade ao choque térmico. A amilase do INPA R020 mostrou maior termoestabilidade a 90ºC e a do INPA R001 a 100˚C. A maior atividade amilolítica mostrada pela amilase do rizóbio INPA R001 foi de 1,6 U/mL e a do rizóbio INPA R020, 7,5 U/mL

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