Gonzalo Durante - Academia.edu (original) (raw)

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Papers by Gonzalo Durante

FEBS Open Bio, 2014

Fructose-1-phosphate (F1P) is the preferred effector of the catabolite repressor/activator (Cra) ... more Fructose-1-phosphate (F1P) is the preferred effector of the catabolite repressor/activator (Cra) protein of the soil bacterium Pseudomonas putida but its ability to bind other metabolic intermediates in vivo is unclear. The Cra protein of this microorganism (Cra PP ) was submitted to mobility shift assays with target DNA sequences (the P fruB promoter) and candidate effectors fructose-1,6-bisphosphate (FBP), glucose 6-phosphate (G6P), and fructose-6-phosphate (F6P). 1 mM F1P was sufficient to release most of the Cra protein from its operators but more than 10 mM of FBP or G6P was required to free the same complex. However, isothermal titration microcalorimetry failed to expose any specific interaction between Cra PP and FBP or G6P. To solve this paradox, transcriptional activity of a P fruB -lacZ fusion was measured in wild-type and DfruB cells growing on substrates that change the intracellular concentrations of F1P and FBP. The data indicated that P fruB activity was stimulated by fructose but not by glucose or succinate. This suggested that Cra PP represses expression in vivo of the cognate fruBKA operon in a fashion dependent just on F1P, ruling out any other physiological effector. Molecular docking and dynamic simulations of the Cra-agonist interaction indicated that both metabolites can bind the repressor, but the breach in the relative affinity of Cra PP for F1P vs FBP is three orders of magnitude larger than the equivalent distance in the Escherichia coli protein. This assigns the Cra protein of P. putida the sole role of transducing the presence of fructose in the medium into a variety of direct and indirect physiological responses.

Systematic and Applied Microbiology, 2013

We studied the possible impact of genomic projects by comparing the number of published articles ... more We studied the possible impact of genomic projects by comparing the number of published articles before and after the completion of the project. We found that for most species, there is no significant change in the number of citations. Also our study remarks the growing importance of taxonomy as main motivation for the sequencing of genomes.

PLoS ONE, 2013

The evolution of transcriptional regulators through the recruitment of DNA-binding domains by enz... more The evolution of transcriptional regulators through the recruitment of DNA-binding domains by enzymes is a widely held notion. However, few experimental approaches have directly addressed this hypothesis. Here we report the reconstruction of a plausible pathway for the evolution of an enzyme into a transcriptional regulator. The BzdR protein is the prototype of a subfamily of prokaryotic transcriptional regulators that controls the expression of genes involved in the anaerobic degradation of benzoate. We have shown that BzdR consists of an N-terminal DNA-binding domain connected through a linker to a C-terminal effector-binding domain that shows significant identity to the shikimate kinase (SK). The construction of active synthetic BzdR-like regulators by fusing the DNA-binding domain of BzdR to the Escherichia coli SKI protein strongly supports the notion that an ancestral SK domain could have been involved in the evolutionary origin of BzdR. The loss of the enzymatic activity of the ancestral SK domain was essential for it to evolve as a regulatory domain in the current BzdR protein. This work also supports the view that enzymes precede the emergence of the regulatory systems that may control their expression.

Journal of Biological Chemistry, 2012

The specific transcriptional regulation of the box pathway for aerobic benzoate degradation is un... more The specific transcriptional regulation of the box pathway for aerobic benzoate degradation is unknown. Results: The BoxR/benzoyl-CoA couple controls the induction of the box genes. Conclusion: BoxR is the regulator of the box pathway in bacteria. Significance: There is cross-regulation between anaerobic and aerobic benzoate degradation pathways.

Journal of Biological Chemistry, 2010

The BzdR transcriptional regulator that controls the P N promoter responsible for the anaerobic c... more The BzdR transcriptional regulator that controls the P N promoter responsible for the anaerobic catabolism of benzoate in Azoarcus sp. CIB constitutes the prototype of a new subfamily of transcriptional regulators. Here, we provide some insights about the functional-structural relationships of the BzdR protein. Analytical ultracentrifugation studies revealed that BzdR is homodimeric in solution. An electron microscopy three-dimensional reconstruction of the BzdR dimer has been obtained, and the predicted structures of the respective N-and C-terminal domains of each BzdR monomer could be fitted into such a reconstruction. Gel retardation and ultracentrifugation experiments have shown that the binding of BzdR to its cognate promoter is cooperative. Different biochemical approaches revealed that the effector molecule benzoyl-CoA induces conformational changes in BzdR without affecting its oligomeric state. The BzdRdependent inhibition of the P N promoter and its activation in the presence of benzoyl-CoA have been established by in vitro transcription assays. The monomeric BzdR4 and BzdR5 mutant regulators revealed that dimerization of BzdR is essential for DNA binding. Remarkably, a BzdR⌬L protein lacking the linker region connecting the N-and C-terminal domains of BzdR is also dimeric and behaves as a super-repressor of the P N promoter. These data suggest that the linker region of BzdR is not essential for protein dimerization, but rather it is required to transfer the conformational changes induced by the benzoyl-CoA to the DNA binding domain leading to the release of the repressor. A model of action of the BzdR regulator has been proposed.

Journal of Bacteriology, 2006

The role of oxygen in the transcriptional regulation of the P N promoter that controls the bzd op... more The role of oxygen in the transcriptional regulation of the P N promoter that controls the bzd operon involved in the anaerobic catabolism of benzoate in the denitrifying Azoarcus sp. strain CIB has been investigated. In vivo experiments using P N ::lacZ translational fusions, in both Azoarcus sp. strain CIB and Escherichia coli cells, have shown an oxygen-dependent repression effect on the transcription of the bzd catabolic genes. E. coli Fnr was required for the anaerobic induction of the P N promoter, and the oxygen-dependent repression of the bzd genes could be bypassed by the expression of a constitutively active Fnr* protein. In vitro experiments revealed that Fnr binds to the P N promoter at a consensus sequence centered at position ؊41.5 from the transcription start site overlapping the ؊35 box, suggesting that P N belongs to the class II Fnr-dependent promoters. Fnr interacts with RNA polymerase (RNAP) and is strictly required for transcription initiation after formation of the RNAP-P N complex. An fnr ortholog, the acpR gene, was identified in the genome of Azoarcus sp. strain CIB. The Azoarcus sp. strain CIB acpR mutant was unable to grow anaerobically on aromatic compounds and it did not drive the expression of the P N ::lacZ fusion, suggesting that AcpR is the cognate transcriptional activator of the P N promoter. Since the lack of AcpR in Azoarcus sp. strain CIB did not affect growth on nonaromatic carbon sources, AcpR can be considered a transcriptional regulator of the Fnr/Crp superfamily that has evolved to specifically control the central pathway for the anaerobic catabolism of aromatic compounds in Azoarcus.

Environmental Microbiology, 2012

Although the genome of Pseudomonas putida KT2440 encodes an orthologue of the crp gene of Escheri... more Although the genome of Pseudomonas putida KT2440 encodes an orthologue of the crp gene of Escherichia coli (encoding the cAMP receptor protein), the regulatory scope of this factor seems to be predominantly co-opted in this bacterium for controlling non-metabolic functions. In order to investigate the reasons for such a functional divergence in otherwise nearly identical proteins, the Crp regulator of P. putida (Crp P. putida) was purified to apparent homogeneity and subject to a battery of in vitro assays aimed at determining its principal physicochemical properties. Analytical ultracentrifugation indicated effector-free Crp P. putida to be a dimer in solution that undergoes a significant change in its hydrodynamic shape in the presence of cAMP. Such a conformational transition was confirmed by limited proteolysis of the protein in the absence or presence of the inducer. Thermodynamic parameters calculated by isothermal titration calorimetry revealed a tight cAMP-Crp P. putida association with an apparent KD of 22.5 Ϯ 2.8 nM, i.e. much greater affinity than that reported for the E. coli's counterpart. The regulator also bound cGMP, but with a K D = 2.6 Ϯ 0.3 mM. An in vitro transcription system was then set up with purified P. putida's RNA polymerase for examining the preservation of the correct protein-protein architecture that makes Crp to activate target promoters. These results, along with cognate gel retardation assays indicated that all basic features of the reference Crp E. coli protein are kept in the P. putida's counterpart, albeit operating under a different set of parameters, the extraordinarily high affinity for cAMP being the most noticeable.