Biochemical and genetic analysis of the Mycobacterium smegmatis CnoX Chaperedoxin
- Authors: Watkins, Ariana Heloise Jo
- Date: 2023-03-29
- Subjects: Mycobacterium smegmatis , Mycobacteria , ENOX1 , Proteostasis , Molecular chaperones , Heat shock proteins , Oxidation-reduction reaction , Redox
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/422403 , vital:71939
- Description: Mycobacterium (M.) tuberculosis (Mtb) encounters numerous physical and chemical stresses associated with host immunity during infection. These include exposure to reactive oxygen, chlorine and nitrogen species, low pH, hypoxia, nutrient starvation, and metal toxicity. Cellular proteins are particularly susceptible to damage by these stresses, and the ability to prevent their irreversible damage is consequently crucial for bacterial growth and survival. Mtb employs a network of proteins that includes chaperones, disaggregases, and proteases to maintain the integrity of its proteome. The chaperedoxin, CnoX, is a recently identified stress-inducible chaperone that combines redox and holdase activities to prevent the over-oxidation and aggregation of proteins in E. coli and other proteobacterial species. In this study, we identified orthologs of the E. coli CnoX (EcCnoX) in Mtb and M. smegmatis (Msm). Bioinformatics analysis of the Mtb and Msm CnoX orthologs (MtCnoX and MsCnoX, respectively) revealed that they possess similar domains, domain architectures and predicted tertiary structures as previously characterised CnoX enzymes, i.e. an N-terminal thioredoxin (Trx) domain fused to a C-terminal TPR-motif containing domain. The EcCnoX, MsCnoX, and MtCnoX enzymes were expressed as recombinant, His-tagged proteins in E. coli and purified to near homogeneity. Biochemical analysis of the recombinant CnoX enzymes revealed that the MsCnoX and MtCnoX both lack thiol-disulphide oxidoreductase (thioredoxin) activity, as evidenced by their inability to catalyse the reduction of the disulphide bonds of insulin in vitro. Both mycobacterial CnoX enzymes displayed activity as chaperones (holdases) during thermal aggregation assays of the model substrate, malate dehydrogenase (MDH). In contrast to previously reported findings for EcCnoX, the holdase activity of the mycobacterial CnoX enzymes was constitutive and did not require exposure to hypochlorous acid (HOCl) for activation. To establish the physiological role of CnoX in Msm, cnoX knockdown (KD) and knockout (KO) mutants were generated using CRISPRi-mediated gene silencing or homologous recombination, respectively. Consistent with previous findings, CnoX activity was not essential for the growth of Msm under conventional growth conditions. Reducing or eliminating CnoX activity in the Msm KD or KO mutants, respectively, did not confer increased sensitivity to HOCl as has been observed for an E. coli cnoX mutant. Reduced CnoX activity in Msm did, however, confer sensitivity to the superoxide generator, plumbagin, and front-line antitubercular drugs rifampicin and isoniazid. The combination of biochemical and physiological data presented suggests that MsCnoX may function as a holdase for substrates following proteotoxic damage induced by certain types of oxidants, a line of investigation that will be pursued in future studies. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2023
- Full Text:
- Date Issued: 2023-03-29
- Authors: Watkins, Ariana Heloise Jo
- Date: 2023-03-29
- Subjects: Mycobacterium smegmatis , Mycobacteria , ENOX1 , Proteostasis , Molecular chaperones , Heat shock proteins , Oxidation-reduction reaction , Redox
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/422403 , vital:71939
- Description: Mycobacterium (M.) tuberculosis (Mtb) encounters numerous physical and chemical stresses associated with host immunity during infection. These include exposure to reactive oxygen, chlorine and nitrogen species, low pH, hypoxia, nutrient starvation, and metal toxicity. Cellular proteins are particularly susceptible to damage by these stresses, and the ability to prevent their irreversible damage is consequently crucial for bacterial growth and survival. Mtb employs a network of proteins that includes chaperones, disaggregases, and proteases to maintain the integrity of its proteome. The chaperedoxin, CnoX, is a recently identified stress-inducible chaperone that combines redox and holdase activities to prevent the over-oxidation and aggregation of proteins in E. coli and other proteobacterial species. In this study, we identified orthologs of the E. coli CnoX (EcCnoX) in Mtb and M. smegmatis (Msm). Bioinformatics analysis of the Mtb and Msm CnoX orthologs (MtCnoX and MsCnoX, respectively) revealed that they possess similar domains, domain architectures and predicted tertiary structures as previously characterised CnoX enzymes, i.e. an N-terminal thioredoxin (Trx) domain fused to a C-terminal TPR-motif containing domain. The EcCnoX, MsCnoX, and MtCnoX enzymes were expressed as recombinant, His-tagged proteins in E. coli and purified to near homogeneity. Biochemical analysis of the recombinant CnoX enzymes revealed that the MsCnoX and MtCnoX both lack thiol-disulphide oxidoreductase (thioredoxin) activity, as evidenced by their inability to catalyse the reduction of the disulphide bonds of insulin in vitro. Both mycobacterial CnoX enzymes displayed activity as chaperones (holdases) during thermal aggregation assays of the model substrate, malate dehydrogenase (MDH). In contrast to previously reported findings for EcCnoX, the holdase activity of the mycobacterial CnoX enzymes was constitutive and did not require exposure to hypochlorous acid (HOCl) for activation. To establish the physiological role of CnoX in Msm, cnoX knockdown (KD) and knockout (KO) mutants were generated using CRISPRi-mediated gene silencing or homologous recombination, respectively. Consistent with previous findings, CnoX activity was not essential for the growth of Msm under conventional growth conditions. Reducing or eliminating CnoX activity in the Msm KD or KO mutants, respectively, did not confer increased sensitivity to HOCl as has been observed for an E. coli cnoX mutant. Reduced CnoX activity in Msm did, however, confer sensitivity to the superoxide generator, plumbagin, and front-line antitubercular drugs rifampicin and isoniazid. The combination of biochemical and physiological data presented suggests that MsCnoX may function as a holdase for substrates following proteotoxic damage induced by certain types of oxidants, a line of investigation that will be pursued in future studies. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2023
- Full Text:
- Date Issued: 2023-03-29
Reduction of tungsten oxides with carbon and hydrogen
- Authors: Venables, Dean Stuart
- Date: 1996
- Subjects: Oxidation-reduction reaction , Tungsten , Hydrogen , Carbon
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4342 , http://hdl.handle.net/10962/d1005004 , Oxidation-reduction reaction , Tungsten , Hydrogen , Carbon
- Description: The reductions of WO₃ with hydrogen, with CO, and with carbon, as well as the reduction of WO₃/graphite mixtures with hydrogen, were studied using thermogravimetry, evolved gas analysis, X-ray powder diffraction, and scanning electron microscopy. The intermediate phases W₂₀O₅₈, W₁₈O₄₉ and WO₂, were observed in the reductions. The final product of the reductions with hydrogen and carbon was tungsten, and we was formed in the reduction with CO. The reaction paths in the overall processes were determined. The reactant/product gas ratio had a considerable influence on which reactions took place. The morphology of the sample was characterised at different stages of the reduction. The shape of the WO₃ particles was retained during the reduction. Particle growth was observed in the reduction with hydrogen and was attributed to the formation of WO₂(OH)₂(g). The kinetics of the reductions were investigated , and the reaction mechanisms determined. The reduction of WO₃ with CO was studied from 650 to 900°C, and occurred at a phase boundary with an activation energy of 40 kJ mol⁻¹ . The reduction of WO₂, was studied under the same conditions. The reaction also occurred at a phase boundary and had an activation energy of 62 kJ mol⁻¹. The reduction of WO₃ with carbon was studied from 935 to 1100°C and took place via CO and CO₂. Two stages were observed in the reduction . The first stage, which corresponded approximately to the formation of WO₂ had an activation energy of 66 kJ mol⁻¹ and was limited by diffusion through the porous reacting particles. The second stage was first order and had an activation energy of 40 kJ mol⁻¹. The reduction of WO₃ and WO₃ graphite mixtures with hydrogen were studied from 575 to 975 °C. The reactions were controlled by mass-transfer under the conditions investigated. The addition of carbon increased the rate of the reduction process , but did not affect the phases formed in the system. CO₂ was evolved mainly at the start, and CO mainly at the end of the process.
- Full Text:
- Date Issued: 1996
- Authors: Venables, Dean Stuart
- Date: 1996
- Subjects: Oxidation-reduction reaction , Tungsten , Hydrogen , Carbon
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4342 , http://hdl.handle.net/10962/d1005004 , Oxidation-reduction reaction , Tungsten , Hydrogen , Carbon
- Description: The reductions of WO₃ with hydrogen, with CO, and with carbon, as well as the reduction of WO₃/graphite mixtures with hydrogen, were studied using thermogravimetry, evolved gas analysis, X-ray powder diffraction, and scanning electron microscopy. The intermediate phases W₂₀O₅₈, W₁₈O₄₉ and WO₂, were observed in the reductions. The final product of the reductions with hydrogen and carbon was tungsten, and we was formed in the reduction with CO. The reaction paths in the overall processes were determined. The reactant/product gas ratio had a considerable influence on which reactions took place. The morphology of the sample was characterised at different stages of the reduction. The shape of the WO₃ particles was retained during the reduction. Particle growth was observed in the reduction with hydrogen and was attributed to the formation of WO₂(OH)₂(g). The kinetics of the reductions were investigated , and the reaction mechanisms determined. The reduction of WO₃ with CO was studied from 650 to 900°C, and occurred at a phase boundary with an activation energy of 40 kJ mol⁻¹ . The reduction of WO₂, was studied under the same conditions. The reaction also occurred at a phase boundary and had an activation energy of 62 kJ mol⁻¹. The reduction of WO₃ with carbon was studied from 935 to 1100°C and took place via CO and CO₂. Two stages were observed in the reduction . The first stage, which corresponded approximately to the formation of WO₂ had an activation energy of 66 kJ mol⁻¹ and was limited by diffusion through the porous reacting particles. The second stage was first order and had an activation energy of 40 kJ mol⁻¹. The reduction of WO₃ and WO₃ graphite mixtures with hydrogen were studied from 575 to 975 °C. The reactions were controlled by mass-transfer under the conditions investigated. The addition of carbon increased the rate of the reduction process , but did not affect the phases formed in the system. CO₂ was evolved mainly at the start, and CO mainly at the end of the process.
- Full Text:
- Date Issued: 1996
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