Jesús Campos-García

Mechanisms of bacterial resistance to chromium compounds

Chromium is a non-essential and well-known toxic metal for microorganisms and plants. The widespread industrial use of this heavy metal has caused it to be considered as a serious environmental pollutant. Chromium exists in nature as two main species, the trivalent form, Cr(III), which is relatively innocuous, and the hexavalent form, Cr(VI), considered a more toxic species. At the intracellular level, however, Cr(III) seems to be responsible for most toxic effects of chromium. Cr(VI) is usually present as the oxyanion chromate. Inhibition of sulfate membrane transport and oxidative damage to biomolecules are associated with the toxic effects of chromate in bacteria. Several bacterial mechanisms of resistance to chromate have been reported. The best characterized mechanisms comprise efflux of chromate ions from the cell cytoplasm and reduction of Cr(VI) to Cr(III). Chromate efflux by the ChrA transporter has been established in Pseudomonas aeruginosa and Cupriavidusmetallidurans (formerly Alcaligenes eutrophus) and consists of an energy-dependent process driven by the membrane potential. The CHR protein family, which includes putative ChrA orthologs, currently contains about 135 sequences from all three domains of life. Chromate reduction is carried out by chromate reductases from diverse bacterial species generating Cr(III) that may be detoxified by other mechanisms. Most characterized enzymes belong to the widespread NAD(P)H-dependent flavoprotein family of reductases. Several examples of bacterial systems protecting from the oxidative stress caused by chromate have been described. Other mechanisms of bacterial resistance to chromate involve the expression of components of the machinery for repair of DNA damage, and systems related to the homeostasis of iron and sulfur.

Transkingdom signaling based on bacterial cyclodipeptides with auxin activity in plants

Microorganisms and their hosts communicate with each other through an array of signals. The plant hormone auxin (indole-3-acetic acid; IAA) is central in many aspects of plant development. Cyclodipeptides and their derivative diketopiperazines (DKPs) constitute a large class of small molecules synthesized by microorganisms with diverse and noteworthy activities. Here, we present genetic, chemical, and plant-growth data showing that in Pseudomonas aeruginosa, the LasI quorum-sensing (QS) system controls the production of three DKPs—namely, cyclo(l-Pro-l-Val), cyclo(l-Pro-l-Phe), and cyclo(l-Pro-l-Tyr)—that are involved in plant growth promotion by this bacterium. Analysis of all three bacterial DKPs in Arabidopsis thaliana seedlings provided detailed information indicative of an auxin-like activity, based on their efficacy at modulating root architecture, activation of auxin-regulated gene expression, and response of auxin-signaling mutants tir1, tir1 afb2 afb3, arf7, arf19, and arf7arf19. The observation that QS-regulated bacterial production of DKPs modulates auxin signaling and plant growth promotion establishes an important function for DKPs mediating prokaryote/eukaryote transkingdom signaling.

Reduction and Efflux of Chromate by Bacteria

The widespread industrial use of chromium has caused this heavy metal to be considered as a serious environmental pollutant. The most common forms of chromium in nature are the relatively innocuous trivalent form, Cr(III), and the more toxic hexavalent species, Cr(VI). Cr(VI) is usually present as the oxyanion chromate. Toxic effects of chromate for bacteria are associated with the inhibition of sulfate transport and with oxidative damage to biomolecules. The best studied bacterial mechanisms of resistance to chromate include reduction of Cr(VI) to the Cr(III) species and efflux of chromate from cell cytoplasm. Several chromate reductases have been identified in diverse bacterial species. Most characterized enzymes belong to the NAD(P)H-dependent flavoprotein family of reductases. Efflux of chromate by the ChrA membrane transporter, a plasmid-encoded protein, has been demonstrated in Pseudomonas and Cupriavidus species. Chromate efflux by ChrA consists of an energy-dependent process driven by the membrane potential. The CHR protein family, which includes putative ChrA homologs, currently contains about 135 sequences from all three domains of life. Other mechanisms of bacterial resistance to chromate involve the expression of components of the machinery for repair of DNA damage as well as free-radical scavenging enzymes.

Ethanol yield and volatile compound content in fermentation of agave must by Kluyveromyces marxianus UMPe-1 comparing with Saccharomyces cerevisiae baker's yeast used in tequila production.

In tequila production, fermentation is an important step. Fermentation determines the ethanol productivity and organoleptic properties of the beverage. In this study, a yeast isolated from native residual agave must was identified as Kluyveromyces marxianus UMPe-1 by 26S rRNA sequencing. This yeast was compared with the bakers yeast Saccharomyces cerevisiae Pan1. Our findings demonstrate that the UMPe-1 yeast was able to support the sugar content of agave must and glucose up to 22% (w/v) and tolerated 10% (v/v) ethanol concentration in the medium with 50% cells survival. Pilot and industrial fermentation of agave must tests showed that the K. marxianus UMPe-1 yeast produced ethanol with yields of 94% and 96% with respect to fermentable sugar content (glucose and fructose, constituting 98%). The S. cerevisiae Pan1 bakers yeast, however, which is commonly used in some tequila factories, showed 76% and 70% yield. At the industrial level, UMPe-1 yeast shows a maximum velocity of fermentable sugar consumption of 2.27g·L(-1)·h(-1) and ethanol production of 1.38g·L(-1)·h(-1), providing 58.78g ethanol·L(-1) at 72h fermentation, which corresponds to 96% yield. In addition, the major and minor volatile compounds in the tequila beverage obtained from UMPe-1 yeast were increased. Importantly, 29 volatile compounds were identified, while the beverage obtained from Pan1-yeast contained fewer compounds and in lower concentrations. The results suggest that the K. marxianus UMPe-1 is a suitable yeast for agave must fermentation, showing high ethanol productivity and increased volatile compound content comparing with a S. cerevisiae bakers yeast used in tequila production.

Phylogenetic analysis of the chromate ion transporter (CHR) superfamily

ChrA is a membrane protein that confers resistance to the toxic ion chromate through the energy‐dependent chromate efflux from the cytoplasm. In the protein databases, ChrA is a member of the chromate ion transporter (CHR) superfamily, composed of at least several dozens of members, distributed in the three domains of life. The aim of this work was to perform a phylogenetic analysis of the CHR superfamily. An exhaustive search for ChrA homologous proteins was carried out at the National Center for Biotechnology Information database. One hundred and thirty‐five sequences were identified as members of the CHR superfamily [77 long‐chain sequences, or bidomains (LCHR), and 58 short‐chain sequences, or monodomains (SCHR)], organized mainly as tandem pairs of genes whose resultant proteins probably possess oppositely oriented membrane topology. LCHR sequences were split into amino and carboxyl domains, and the resultant domains were aligned with the SCHR proteins. A phylogenetic tree was reconstructed using four different methods, obtaining similar results. The domains were grouped into three clusters: the SCHR proteins cluster, the amino domain cluster of LCHR proteins and the carboxyl domain cluster of LCHR proteins. These results, as well as differences in the genomic context of CHR proteins, enabled the proteins to be sorted into two families (SCHR and LCHR), and 10 subfamilies. Evidence was found suggesting an ancient origin of LCHR proteins from the fusion of two SCHR protein‐encoding genes; however, some secondary events of fusion and fission may have occurred later. The separate distribution of the LCHR and SCHR proteins, differences in the genomic context in both groups and the fact that chromate transport has been demonstrated only in LCHR proteins suggest that the CHR proteins comprise two or more paralogous groups in the CHR superfamily.

Reactive oxygen species production induced by ethanol in Saccharomyces cerevisiae increases because of a dysfunctional mitochondrial iron–sulfur cluster assembly system

Ethanol accumulation during fermentation contributes to the toxic effects in Saccharomyces cerevisiae, impairing its viability and fermentative capabilities. The iron-sulfur (Fe-S) cluster biogenesis is encoded by the ISC genes. Reactive oxygen species (ROS) generation is associated with iron release from Fe-S-containing enzymes. We evaluated ethanol toxicity, ROS generation, antioxidant response and mitochondrial integrity in S. cerevisiae ISC mutants. These mutants showed an impaired tolerance to ethanol. ROS generation increased substantially when ethanol accumulated at toxic concentrations under the fermentation process. At the cellular and mitochondrial levels, ROS were increased in yeast treated with ethanol and increased to a higher level in the ssq1∆, isa1∆, iba57∆ and grx5∆ mutants - hydrogen peroxide and superoxide were the main molecules detected. Additionally, ethanol treatment decreased GSH/GSSG ratio and increased catalase activity in the ISC mutants. Examination of cytochrome c integrity indicated that mitochondrial apoptosis was triggered following ethanol treatment. The findings indicate that the mechanism of ethanol toxicity occurs via ROS generation dependent on ISC assembly system functionality. In addition, mutations in the ISC genes in S. cerevisiae contribute to the increase in ROS concentration at the mitochondrial and cellular level, leading to depletion of the antioxidant responses and finally to mitochondrial apoptosis.

Essential residues in the chromate transporter ChrA of Pseudomonas aeruginosa

The chrA gene of Pseudomonas aeruginosa plasmid pUM505 encodes the hydrophobic protein ChrA, which confers resistance to chromate by the energy-dependent efflux of chromate ions. Chromate-sensitive mutants were isolated by in vivo random mutagenesis. Transport experiments with cell suspensions of selected mutants showed that 51CrO4(2-) extrusion was drastically lowered as compared to suspensions of the strain with the wild-type plasmid, confirming that the mutations affected a chromate efflux system. DNA sequence analysis showed that most point mutations affected amino acids clustered in the N-terminal half of ChrA, altering either cytoplasmic regions or transmembrane segments, and replaced residues moderately to highly conserved in ChrA homologs. PhoA and LacZ translational fusions were used to confirm the membrane topology at the N-terminal half of the ChrA protein.

Effects of diabetes on oxidative and nitrosative stress in kidney mitochondria from aged rats

Diabetes mellitus (DM) is characterized by chronic hyperglycemia resulting from defects in the secretion and/or action of insulin. Diabetic nephropathy (DN) develops in diabetic patients and is characterized by a progressive deterioration of renal function. The mitochondrial electron transport chain (ETC) produces most of the reactive oxygen species (ROS) that are involved in diabetic nephropathy. Due to the high incidence of DM in the elderly, the aim of this study was to evaluate oxidative and nitrosative stress in kidney mitochondria from aged rats. We evaluated lipid peroxidation (LPO), nitric oxide (NO•) production, S-nitrosylation profiles, glutathione levels, and glutathione reductase and aconitase activities under streptozotocin (STZ)-induced experimental diabetes in kidney mitochondria from aged rats. The results showed an increase in LPO, NO• production, and S-nitrosylated proteins in rats with STZ-induced diabetes. A decrease in glutathione (GSH) levels and glutathione reductase (GR) and aconitase activities in the rats that received the STZ-induced diabetes treatment was also observed, when compared with the age-related controls. The data suggest that oxidative and nitrosative stresses promote mitochondrial oxidative dysfunction in the more advanced age rat kidney in STZ-induced diabetes.

Protective effects of dietary avocado oil on impaired electron transport chain function and exacerbated oxidative stress in liver mitochondria from diabetic rats.

Electron transport chain (ETC) dysfunction, excessive ROS generation and lipid peroxidation are hallmarks of mitochondrial injury in the diabetic liver, with these alterations also playing a role in the development of non-alcoholic fatty liver disease (NAFLD). Enhanced mitochondrial sensitivity to lipid peroxidation during diabetes has been also associated to augmented content of C22:6 in membrane phospholipids. Thus, we aimed to test whether avocado oil, a rich source of C18:1 and antioxidants, attenuates the deleterious effects of diabetes on oxidative status of liver mitochondria by decreasing unsaturation of acyl chains of membrane lipids and/or by improving ETC functionality and decreasing ROS generation. Streptozocin-induced diabetes elicited a noticeable increase in the content of C22:6, leading to augmented mitochondrial peroxidizability index and higher levels of lipid peroxidation. Mitochondrial respiration and complex I activity were impaired in diabetic rats with a concomitant increase in ROS generation using a complex I substrate. This was associated to a more oxidized state of glutathione, All these alterations were prevented by avocado oil except by the changes in mitochondrial fatty acid composition. Avocado oil did not prevented hyperglycemia and polyphagia although did normalized hyperlipidemia. Neither diabetes nor avocado oil induced steatosis. These results suggest that avocado oil improves mitochondrial ETC function by attenuating the deleterious effects of oxidative stress in the liver of diabetic rats independently of a hypoglycemic effect or by modifying the fatty acid composition of mitochondrial membranes. These findings might have also significant implications in the progression of NAFLD in experimental models of steatosis.

Malfunctioning of the Iron-Sulfur Cluster Assembly Machinery in Saccharomyces cerevisiae Produces Oxidative Stress via an Iron-Dependent Mechanism, Causing Dysfunction in Respiratory Complexes

Biogenesis and recycling of iron–sulfur (Fe–S) clusters play important roles in the iron homeostasis mechanisms involved in mitochondrial function. In Saccharomyces cerevisiae, the Fe–S clusters are assembled into apoproteins by the iron–sulfur cluster machinery (ISC). The aim of the present study was to determine the effects of ISC gene deletion and consequent iron release under oxidative stress conditions on mitochondrial functionality in S. cerevisiae. Reactive oxygen species (ROS) generation, caused by H2O2, menadione, or ethanol, was associated with a loss of iron homeostasis and exacerbated by ISC system dysfunction. ISC mutants showed increased free Fe2+ content, exacerbated by ROS-inducers, causing an increase in ROS, which was decreased by the addition of an iron chelator. Our study suggests that the increment in free Fe2+ associated with ROS generation may have originated from mitochondria, probably Fe–S cluster proteins, under both normal and oxidative stress conditions, suggesting that Fe–S cluster anabolism is affected. Raman spectroscopy analysis and immunoblotting indicated that in mitochondria from SSQ1 and ISA1 mutants, the content of [Fe–S] centers was decreased, as was formation of Rieske protein-dependent supercomplex III2IV2, but this was not observed in the iron-deficient ATX1 and MRS4 mutants. In addition, the activity of complexes II and IV from the electron transport chain (ETC) was impaired or totally abolished in SSQ1 and ISA1 mutants. These results confirm that the ISC system plays important roles in iron homeostasis, ROS stress, and in assembly of supercomplexes III2IV2 and III2IV1, thus affecting the functionality of the respiratory chain.