Roberto Velasco-García

Continuous Cr(VI) removal by Scenedesmus incrassatulus in an airlift photobioreactor

Cr(VI) removal by Scenedesmus incrassatulus was characterized in a continuous culture system using a split-cylinder internal-loop airlift photobioreactor fed continuously with a synthetic effluent containing 1.0mg Cr(VI) l(-1) at dilution rate (D) of 0.3d (-1). At steady state, there was a small increase (6%) on the dry biomass (DB) concentration of Cr(VI)-treated cultures compared with the control culture. 1.0mg Cr(VI) l(-1) reduced the photosynthetic pigments content and altered the cellular morphology, the gain in dry weight was not affected. At steady state, Cr(VI) removal efficiency was 43.5+/-1.0% and Cr(VI) uptake was 1.7+/-0.1 mg Cr(VI) g(-1) DB. The system reached a specific metal removal rate of 458 microg Cr(VI) g(-1) DB d(-1), and a volumetric removal rate of 132 microg Cr(VI) l(-1) d(-1).

Betaine aldehyde dehydrogenase from Pseudomonas aeruginosa: cloning, over-expression in Escherichia coli, and regulation by choline and salt

In the human pathogen Pseudomonas aeruginosa, betaine aldehyde dehydrogenase (BADH) may play a dual role assimilating carbon and nitrogen from choline or choline precursors—abundant at infection sites—and producing glycine betaine, which protects the bacteria against the high-osmolarity stress prevalent in the infected tissues. We cloned the P. aeruginosa BADH gene and expressed the BADH protein in Escherichia coli. The recombinant protein appears identical to its native counterpart, as judged by Western blot, N-terminal amino acid sequence, tryptophan-fluorescence emission spectra, circular-dichroism spectroscopy, size-exclusion chromatography, and kinetic properties. Computational analysis indicated that the promoter sequence of the putative operon that includes the BADH gene has a consensus-binding site for the choline-sensing transcription repressor BetI, and putative boxes for ArcA and Lrp transcription factors but no known elements of response to osmotic stress. This is consistent with the strong induction of BADH expression by choline and with the lack of effect of NaCl. As there were significant amounts of BADH protein and activity in P. aeruginosa cells grown on glucose plus choline, as well as the BADH activity exhibiting tolerance to salt, it is likely that glycine betaine is synthesized in vivo and could play an important osmoprotectant role under conditions of infection.

Monovalent cations requirements for the stability of betaine aldehyde dehydrogenase from Pseudomonas aeruginosa, porcine kidney and amaranth leaves

Betaine aldehyde dehydrogenase from the human pathogen Pseudomonas aeruginosa requires K(+) ions for maintenance of its active conformation. In order to explore if this property is shared by other BADHs of different origins and to further understand the mechanism underlying the effects of these ions, we carried out a comparative study on the stability and quaternary structure of P. aeruginosa, porcine kidney and amaranth leaves BADHs in the absence of K(+) ions. At low enzyme concentrations, the bacterial and porcine enzymes were totally inactivated upon removal of K(+) following biphasic and monophasic kinetics, respectively, whereas the amaranth enzyme retained its activity. Inactivation of P. aeruginosa BADH was much faster than that of the porcine enzyme. The oxidized coenzyme protected both enzymes against inactivation by the absence of K(+), whereas betaine aldehyde afforded partial protection to the bacterial BADH and increased the inactivation rate of the porcine. Reactivation of the inactive enzymes, by adding back to the incubation medium K(+) ions, was dependent on enzyme concentration, suggesting that enzyme dissociation takes place in the absence of K(+). In the bacterial enzyme, NH(4)(+) but not Na(+) ions could mimic the effects of K(+), whereas the three cations tested reactivated porcine BADH, indicating a requirement of this enzyme for high ionic strength rather than for a specific monovalent cation. Size exclusion chromatography of the inactivated enzymes confirmed that K(+) ions or other monovalent cations are required for the maintenance of the quaternary structure of these two BADHs. At pH 7.0, in the absence of K(+) in a buffer of low ionic strength, the active tetrameric form of P. aeruginosa BADH dissociated into inactive monomers and that of porcine kidney BADH into inactive dimers. Once reactivated, both enzymes reassociated into active tetramers.

Inactivation of betaine aldehyde dehydrogenase from Pseudomonas aeruginosa and Amaranthus hypochondriacus L. leaves by disulfiram.

Betaine aldehyde dehydrogenase (BADH) activity might be crucial for the growth of the human pathogen Pseudomonas aeruginosa under conditions of infection and therefore appears to be a suitable target for antimicrobial agents. As a first step in the search for BADH inhibitors, we have tested the effects of the known aldehyde dehydrogenase inhibitor disulfiram (DSF) on the activity of P. aeruginosa and Amaranthus hypochondriacus (amaranth) leaf BADHs. DSF totally inactivated both enzymes in a time- and dose-dependent manner. In the case of the Pseudomonas enzyme, inactivation kinetics were monophasic with a second-order inactivation rate constant at pH 6.9 of 4.9+/-0.4 M(-1) s(-1), whereas the plant enzyme was inactivated in a biphasic process with second-order inactivation rate constants at pH 7.5 of 6.8+/-0.6 and 0.33+/-0.04 M(-1) s(-1). At pH 8.8, the second-order rate constants for inactivation of the bacterial enzyme was 1 x 10(3) M(-1) s(-1), which compare well with that reported for human liver mitochondrial aldehyde dehydrogenase (ALDH2), the target of DSF inhibition in the aversion therapy of alcoholism. Both BADHs were inactivated faster in the presence of NAD(P)(+) than in its absence, whereas NAD(P)H and betaine aldehyde protected the bacterial, but increased the inactivation rate of the plant enzyme. The inactivated enzymes were reactivated by dithiothreitol, but not by a high concentration of the physiological reductant glutathione. The high in vitro sensitivity of the Pseudomonas BADH to DSF, particularly in the presence of NAD(P)(+), together with the lack of reversibility of DSF modification by glutathione, makes this inhibitor a potential antimicrobial agent and suggests that it might be worth testing its effects and those of its metabolites in vivo, under culture conditions in which the activity of BADH is required for growth of the bacteria.

The study of protein-protein interactions in bacteria.

Many of the functions fulfilled by proteins in the cell require specific protein-protein interactions (PPI). During the last decade, the use of high-throughput experimental technologies, primarily based on the yeast 2-hybrid system, generated extensive data currently located in public databases. This information has been used to build interaction networks for different species. Unfortunately, due to the nature of the yeast 2-hybrid system, these databases contain many false positives and negatives, thus they require purging. A method for confirming these PPI is to test them using a technique that operates in vivo and detects binary PPI. This article comprises an overview of the study of PPI and describes the main techniques that have been used to identify bacterial PPI, prioritizing those that can be used for their verification, and it also mentions a number of PPI that have been identified or confirmed using these methods.

Identification of hepatic protein-protein interaction targets for betaine homocysteine S-methyltransferase

Protein-protein interactions are an important mechanism for the regulation of enzyme function allowing metabolite channeling, crosstalk between pathways or the introduction of post-translational modifications. Therefore, a number of high-throughput studies have been carried out to shed light on the protein networks established under different pathophysiological settings. Surprisingly, this type of information is quite limited for enzymes of intermediary metabolism such as betaine homocysteine S-methyltransferase, despite its high hepatic abundancy and its role in homocysteine metabolism. Here, we have taken advantage of two approaches, affinity purification combined with mass spectrometry and yeast two-hybrid, to further uncover the array of interactions of betaine homocysteine S-methyltransferase in normal liver of Rattus norvegicus. A total of 131 non-redundant putative interaction targets were identified, out of which 20 were selected for further validation by coimmunoprecipitation. Interaction targets validated by two different methods include: S-methylmethionine homocysteine methyltransferase or betaine homocysteine methyltransferase 2, methionine adenosyltransferases α1 and α2, cAMP-dependent protein kinase catalytic subunit alpha, 4-hydroxyphenylpyruvic acid dioxygenase and aldolase b. Network analysis identified 122 nodes and 165 edges, as well as a limited number of KEGG pathways that comprise: the biosynthesis of amino acids, cysteine and methionine metabolism, the spliceosome and metabolic pathways. These results further expand the connections within the hepatic methionine cycle and suggest putative cross-talks with additional metabolic pathways that deserve additional research.