Papers by Author: Carlos A. Jerez

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Authors: Carlos A. Jerez
Abstract: Systems Microbiology is a new way to approach research in microbiology. The idea is to treat the microorganism or community as a whole, integrating fundamental biological knowledge with OMICS research (genomics, proteomics, transcriptomics, metabolomics) and bioinformatics to obtain a global picture of how a microbial cell operates in the community. The oxidative reactions resulting in the extraction of dissolved metal values from ores is the outcome of a consortium of different microorganisms. Therefore, this bioleaching community is particularly amenable for the application of Systems Microbiology. As more genomic sequences of different biomining microorganisms become available, it will be possible to define the molecular adaptations of bacteria to their environment, the interactions between the members of the community and to predict favorable or negative changes to efficiently control metal solubilization. Some key phenomena to understand the process of biomining are biochemistry of iron and sulfur compound oxidation, bacteria-mineral interactions (chemotaxis, cell-cell communication, adhesion, biofilm formation) and several adaptive responses allowing the microorganisms to survive in a bioleaching environment. These variables should be considered in an integrative way from now on. Together with recently developed molecular methods to monitor the behavior and evolution of microbial participants during bioleaching operations, Systems Microbiology will offer a comprehensive view of the bioleaching community. The power of the OMICS approaches will be briefly reviewed. It is expected they will provide not only exciting new findings but also will allow predictions on how to keep the microbial consortium healthy and therefore efficient during the entire process of bioleaching.
Authors: Lina María Ruíz, Wolfgang Sand, Carlos A. Jerez, Nicolas Guiliani
Abstract: Acidithiobacillus ferrooxidans, an acidophilic, chemolithotrophic, γ-proteobacterium, is involved in the bioleaching of metal sulfides. For this process, bacterial attachment to mineral surface and biofilm development play a pivotal role. Generally, biofilm formation and production of exopolysaccharides is regulated by the second messenger cyclic diguanylic acid (c-di-GMP) whose cellular level depends on the synthesis and degradation activities of diguanylate cyclase (DGCs, with GGDEF domain) and phosphodiesterase (PDE, with EAL or HD-GYP domains), respectively. The analysis of the genomic sequence of A. ferrooxidans ATCC 23270 allowed us to identify 5 putative orfs encoding DGC and/or PDE-like proteins. Four of them encode for bifunctional putative proteins with GGDEF and EAL domains and are named AFE_0053, AFE_1360, AFE_1373 and AFE_1379. The fifth one named AFE_1852 has an EAL domain. The putative proteins also include PAS and GAF domains involved in signal transduction. These features suggest an involvement in signalling transduction through the metabolism of c-di-GMP. The amino acid sequences of these putative proteins were aligned with known DGCs and PDEs. Alignments indicate that AFE_1360 and AFE_1373 share more consensus sequences with active PDEs, whereas AFE_0053 and AFE_1379 do with active DGCs. On the other hand, in AFE_1852 some conserved residues of known active PDEs are changed. RT-PCR-experiments revealed that the genes that encode for these putative DGCs and/or PDEs are expressed by growth on two different substrates. These preliminary results suggest that A. ferrooxidans possesses a c-di-GMP pathway that should be involved in biofilm formation, as it occurs in many bacteria.
Authors: M. Castro, Lina María Ruíz, A. Barriga, Carlos A. Jerez, David S. Holmes, Nicolas Guiliani
Abstract: Acidithiobacillus ferrooxidans, A. thiooxidans, and A. caldus are acidophilic Gram-negative -proteobacteria involved in the bioleaching of metal sulfides. Bacterial attachment to mineral surface and biofilm development play a pivotal role in this process. Therefore, the understanding of biofilm formation has relevance to the design of biological strategies to improve the efficiency of bioleaching processes. For this reason, our laboratory is focused on the characterization of the molecular mechanisms involved in biofilm formation in biomining bacteria. In many bacteria, the intracellular level of c-di-GMP molecules regulates the transition from the motile planktonic state to sessile community-based behaviors, such as biofilm development. Thus, we recently started the study of c-di-GMP pathway in biomining bacteria. C-di-GMP molecules are synthesized by diguanylate cyclases (DGCs) and degraded by phosphodiesterases (PDEs). So far, two kinds of effectors have been identified, including three protein families (pilZ, PleD and FleQ) and a conserved RNA domain (GEMM) which acts as a riboswitch. We previously reported the existence of different molecular players involved in c-di-GMP pathway in A. ferrooxidans ATCC 23270. Here, we expanded our work to other Acidithioibacillus species: A. thiooxidans ATCC 19377 and A. caldus ATCC 51756. In both, we identified several putative-ORFs encoding DGC, PDE and effector proteins. By using total RNA extracted from A. ferrooxidans and A. caldus cells in RT-PCR and qPCR experiments, we demonstrated that these genes are expressed. In addition, we characterized the presence of c-di-GMP in A. ferrooxidans ATCC 23270 and A. caldus ATCC 51756 cell extracts. Taken together, these results strongly suggest that A. ferrooxidans, A. caldus and A. thiooxidans possess functional c-di-GMP pathways. As it occurs in other Gram-negative bacteria, this pathway should be involved in the regulation of the planktonic/biofilm switch. In the future, we have to integrate this new biological dimension to improve the biological understanding of bioleaching.
Authors: A. Orell, C.A. Navarro, Carlos A. Jerez
Abstract: Extremophiles such as the acidophilic Sulfolobus metallicus (Archaea) and Acidithiobacillus ferrooxidans (Bacteria) can resist Cu (CuSO4) concentrations of 200 mM and 800 mM respectively. These microorganisms are important in biomining processes to extract copper and other metals. A. ferrooxidans grown at low Cu concentrations (5 mM) expressed genes coding for ATPases most likely involved in pumping the metal from the cytoplasm to the periplasm of the bacterium. At 100 mM Cu the previous systems were repressed and there was a great induction in the expression of efflux systems known to use the proton motive force energy to export the metal outside the cell. These Cu-resistance determinants from A. ferrooxidans were found to be functional since when expressed in Escherichia coli they conferred higher Cu tolerance to it. Novel Cu-resistance determinants for A. ferrooxidans were found and characterized. S. metallicus possessed at least 2 CopM metallochaperones and 2 CopA ATPases whose expressions were induced by Cu (5 to 50 mM). Furthermore, we previously reported that both microorganisms accumulate high levels of inorganic polyphosphate (PolyP) and that intracellular Cu concentration stimulates polyP hydrolysis. The resulting Pi would then be transported out of the cell as a metal-Pi complex to detoxify the cells. In addition, our results suggest that at high Cu concentrations polyP could also provide energy for the metal efflux. All the data suggest that both biomining microorganisms use different systems to respond to Cu depending on the extracellular concentrations of the metal and suggest that the presence of different additional systems to respond to Cu may explain the extremely high metal resistance of these extremophiles.
Authors: Lina María Ruíz, Alex Gonzalez, Marine Frezza, Laurent Soulère, Yves Queneau, Alain Doutheau, Thore Rohwerder, Wolfgang Sand, Carlos A. Jerez, Nicolas Guiliani
Abstract: Biofilm development plays a pivotal role in the bioleaching process. The attachment of the acidophilic chemolithotrophic Acidithiobacillus ferrooxidans to mineral surfaces is mediated by extracellular polymeric substances (EPS) involved in biofilm development. Previous work suggests that EPS composition of A. ferrooxidans is adapted to the energy source and, accordingly, the bacterium must be able to sense the surface to which attachment occurs with the consequent triggering of the expression of different EPS-genes. Quorum sensing (QS) is recognized as one of the main regulators of biofilm formation. A. ferrooxidans possesses a functional QS type AI-1 system and the analysis of culture supernatants revealed us that this bacterium is able to synthesize nine different homoserine lactones (AHLs) whose acyl-chain lengths oscillate between 8 and 16 carbons and include an alcohol or a ketone function at the C3 position. The transcription levels of the afeI gene encoding for the AHL synthase are higher in cells grown in sulfur and thiosulfate media than in iron-grown cells, suggesting that biofilm formation in A. ferrooxidans would be regulated by the QS type AI-1 system. In the present study, the effect of several synthetic AHLs and analogues on the attachment of A. ferrooxidans to pyrite was analyzed. Preliminary results suggest that some of these molecules are changing the bacterial attachment to pyrite.
Authors: Rodrigo Almarcegui, Claudio Navarro, Alberto Paradela, Diego von Bernath, Carlos A. Jerez
Abstract: The presence in At. ferrooxidans of canonical copper resistance determinants does not explain the extremely high copper concentrations this microorganism is able to tolerate. This suggests that At. ferrooxidans may have additional copper resistance mechanisms. New possible copper resistance determinants were searched by using 2D-PAGE and real time PCR (qRT-PCR). Results showed the up-regulation of RND-type Cus systems and different RND-type efflux pumps in At. ferrooxidans grown in the presence of copper, suggesting that these proteins may be implied in resistance to this metal. Furthermore, the up-regulation of putative periplasmatic disulfide isomerases was also seen in the presence of copper. These proteins are most likely involved in the formation and rearrangement of disulfide bonds in proteins in the periplasm. Copper ions catalyze the formation of incorrect disulfide bonds in proteins. However, the up-regulated disulfide isomerases found could restore native disufide bonds allowing cell survival. In conclusion, At. ferrooxidans may resist high copper concentrations by using additional copper resistance strategies in which cell envelope proteins are very important. This knowledge could be used to select the best fit members of the bioleaching community to attain more efficient industrial biomining processes.
Authors: Alex Gonzalez, María José Gallardo, Marine Frezza, Laurent Soulère, Yves Queneau, Alain Doutheau, Carlos A. Jerez, Nicolas Guiliani
Authors: F.A. Pagliai, Carlos A. Jerez
Abstract: Acidithiobacillus ferrooxidans is a chemolithoautotrophic acidophile capable of catalyzing the oxidation of ferrous iron and sulfur reducing compounds. During the dissolution of ores A. ferrooxidans adheres to the solid substrate by hydrophobic and hydrophilic interactions and also extracellular polymeric substances may be involved. Nevertheless, many of these agents that participate in the process are unknown. Therefore, the identification and characterization of the extracellular proteome of A. ferrooxidans was addressed during this work. First, by searching the genome of A. ferrooxidans ATCC 23270 the putative genes coding for proteins present in most of the different secretion systems (type I, II, IV, V systems and the Usher pathway) were found. Second, when the secretion signal prediction server SubCel 1.0 was used, 26.7 % of the total open reading frames showed possible signals that would allow these proteins to leave the cytoplasm towards the internal and outer membranes, the periplasm, or the extracellular milieu. By using 2D-NEPHGE and MS sequencing we identified the eleven most abundant proteins present in the extracellular fraction. Proteins related to the transport and binding of solutes and the folding of proteins, among others were identified. Along with the non-classical pathway, the protein secretion systems identified could constitute the secretion machinery for the proteins found in the extracellular sub-proteome of A. ferrooxidans that as a whole constitute its secretome. Further studies of the extracellular proteins from this biomining microorganism will be important to find out their possible role if any, during bacteria-mineral interactions.
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