Publicação
Study of a new pathway involved in electron bifurcation in araerobic bacteria
| Resumo: | Bioenergetics of chemotrophic bacteria is based on substrate-level phosphorylation and electron transfer phosphorylation for energy conservation. Recently, a third mechanism of energy coupling named flavin-based electron bifurcation (FBEB) was proposed for anaerobic bacteria. Sulfate-reducing organisms (SRO) are a polyphyletic group of anaerobic microorganisms, which perform dissimilatory sulfate reduction. Interestingly, studies concerning SRO identified protein homologies to enzymes engaged in FBEB in methanogens, suggesting the presence of FBEB in sulfate reducers. Here we studied: i) the physiological role of a protein complex (HdrABC-FloxABCD) possibly involved in FBEB; and ii) the function of DsrD, a protein potentially involved in sulfite reduction, the last step of the dissimilatory sulfate reduction. Firstly, phenotypic characterization of hdrC and floxA mutants in Desulfovibrio vulgaris Hildenborough with ethanol as electron donor revealed no cell growth, while a complemented strain of the floxA mutant grew similarly to the wild-type (WT). Then, under pyruvate fermentation conditions, both mutants produced low levels of ethanol comparing to the WT and complemented strain. Gene and protein expression analysis in WT strains cultured with different electron donors/acceptors showed upregulation of FloxA and HdrA when ethanol is the electron donor. Moreover, an alcohol dehydrogenase (adh1) gene present upstream of the hdr-flox cluster is also upregulated in the medium containing ethanol. Altogether, these results show that the HdrABC-FloxABCD complex is involved in the ethanol metabolism of D. vulgaris. Secondly, the dsrD gene from D. vulgaris was cloned, overexpressed and the protein purified. Sulfite reduction activity and protein-protein interaction studies showed no direct biochemical role of DsrD in sulfite reduction. Then a dsrD deletion mutant was generated showing a long lag phase under sulfate reduction conditions when compared to the WT. This mutant did not grow with sulfite as electron acceptor, revealing the importance of dsrD in sulfite reduction, most likely at a regulatory level. Overall, this work allowed a better understanding of energy conservation mechanisms in SRO, proposing a new mechanism for ethanol metabolism played by the FloxABCD-HdrABC complex and producing new insights into the function of DsrD in the dissimilatory sulfate reduction. |
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| Autores principais: | Oliveira, Gonçalo Pizarro Madureira Salgado de, 1991- |
| Assunto: | Bioenergia Bactérias anaeróbicas Expressão génica Teses de mestrado - 2014 |
| Ano: | 2014 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | Bioenergetics of chemotrophic bacteria is based on substrate-level phosphorylation and electron transfer phosphorylation for energy conservation. Recently, a third mechanism of energy coupling named flavin-based electron bifurcation (FBEB) was proposed for anaerobic bacteria. Sulfate-reducing organisms (SRO) are a polyphyletic group of anaerobic microorganisms, which perform dissimilatory sulfate reduction. Interestingly, studies concerning SRO identified protein homologies to enzymes engaged in FBEB in methanogens, suggesting the presence of FBEB in sulfate reducers. Here we studied: i) the physiological role of a protein complex (HdrABC-FloxABCD) possibly involved in FBEB; and ii) the function of DsrD, a protein potentially involved in sulfite reduction, the last step of the dissimilatory sulfate reduction. Firstly, phenotypic characterization of hdrC and floxA mutants in Desulfovibrio vulgaris Hildenborough with ethanol as electron donor revealed no cell growth, while a complemented strain of the floxA mutant grew similarly to the wild-type (WT). Then, under pyruvate fermentation conditions, both mutants produced low levels of ethanol comparing to the WT and complemented strain. Gene and protein expression analysis in WT strains cultured with different electron donors/acceptors showed upregulation of FloxA and HdrA when ethanol is the electron donor. Moreover, an alcohol dehydrogenase (adh1) gene present upstream of the hdr-flox cluster is also upregulated in the medium containing ethanol. Altogether, these results show that the HdrABC-FloxABCD complex is involved in the ethanol metabolism of D. vulgaris. Secondly, the dsrD gene from D. vulgaris was cloned, overexpressed and the protein purified. Sulfite reduction activity and protein-protein interaction studies showed no direct biochemical role of DsrD in sulfite reduction. Then a dsrD deletion mutant was generated showing a long lag phase under sulfate reduction conditions when compared to the WT. This mutant did not grow with sulfite as electron acceptor, revealing the importance of dsrD in sulfite reduction, most likely at a regulatory level. Overall, this work allowed a better understanding of energy conservation mechanisms in SRO, proposing a new mechanism for ethanol metabolism played by the FloxABCD-HdrABC complex and producing new insights into the function of DsrD in the dissimilatory sulfate reduction. |
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