Examinando por Autor "Esparza, Mario"
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Ítem Expression and activity of the Calvin-Benson-Bassham cycle transcriptional regulator CbbR from Acidithiobacillus ferrooxidans in Ralstonia eutropha(Oxford University Press, 2015-08) Esparza, Mario; Jedlicki, Eugenia; Dopson, Mark; Holmes, David S.Autotrophic fixation of carbon dioxide into cellular carbon occurs via several pathways but quantitatively, the Calvin-Benson-Bassham cycle is the most important. CbbR regulates the expression of the cbb genes involved in CO2 fixation via the Calvin-Benson-Bassham cycle in a number of autotrophic bacteria. A gene potentially encoding CbbR (cbbRAF) has been predicted in the genome of the chemolithoautotrophic, extreme acidophile Acidithiobacillus ferrooxidans. However, this microorganism is recalcitrant to genetic manipulation impeding the experimental validation of bioinformatic predictions. Two novel functional assays were devised to advance our understanding of cbbRAF function using the mutated facultative autotroph Ralstonia eutropha H14 ΔcbbR as a surrogate host to test gene function: (i) cbbRAF was expressed in R. eutropha and was able to complement ΔcbbR; and (ii) CbbRAF was able to regulate the in vivo activity of four A. ferrooxidans cbb operon promoters in R. eutropha. These results open up the use of R. eutropha as a surrogate host to explore cbbRAF activity. © FEMS 2015.Ítem Genes and pathways for CO2fixation in the obligate, chemolithoautotrophic acidophile, Acidithiobacillus ferrooxidans, Carbon fixation in A. ferrooxidans(BMC, 2010) Esparza, Mario; Crdenas, Juan Pablo; Bowien, Botho; Jedlicki, Eugenia; Holmes, David S.Background. Acidithiobacillus ferrooxidans is chemolithoautotrophic -proteobacterium that thrives at extremely low pH (pH 1-2). Although a substantial amount of information is available regarding CO2uptake and fixation in a variety of facultative autotrophs, less is known about the processes in obligate autotrophs, especially those living in extremely acidic conditions, prompting the present study. Results. Four gene clusters (termed cbb1-4) in the A. ferrooxidans genome are predicted to encode enzymes and structural proteins involved in carbon assimilation via the Calvin-Benson- Bassham (CBB) cycle including form I of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO, EC 4.1.1.39) and the CO2- concentrating carboxysomes. RT-PCR experiments demonstrated that each gene cluster is a single transcriptional unit and thus is an operon. Operon cbb1 is divergently transcribed from a gene, cbbR, encoding the LysR-type transcriptional regulator CbbR that has been shown in many organisms to regulate the expression of RubisCO genes. Sigma70-like -10 and -35 promoter boxes and potential CbbR-binding sites (T-N11-A/TNA-N7TNA) were predicted in the upstream regions of the four operons. Electrophoretic mobility shift assays (EMSAs) confirmed that purified CbbR is able to bind to the upstream regions of the cbb1, cbb2 and cbb3 operons, demonstrating that the predicted CbbR-binding sites are functional in vitro. However, CbbR failed to bind the upstream region of the cbb4 operon that contains cbbP, encoding phosphoribulokinase (EC 2.7.1.19). Thus, other factors not present in the assay may be required for binding or the region lacks a functional CbbR-binding site. The cbb3 operon contains genes predicted to encode anthranilate synthase components I and II, catalyzing the formation of anthranilate and pyruvate from chorismate. This suggests a novel regulatory connection between CO 2fixation and tryptophan biosynthesis. The presence of a form II RubisCO could promote the ability of A. ferrooxidans to fix CO2at different concentrations of CO2. Conclusions. A. ferrooxidans has features of cbb gene organization for CO2-assimilating functions that are characteristic of obligate chemolithoautotrophs and distinguish this group from facultative autotrophs. The most conspicuous difference is a separate operon for the cbbP gene. It is hypothesized that this organization may provide greater flexibility in the regulation of expression of genes involved in inorganic carbon assimilation. © 2010 Esparza et al; licensee BioMed Central Ltd.