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Invasive Pulmonary Aspergillosis, which is caused by the filamentous fungus Aspergillus fumigatus, is a life-threatening infection for immunosuppressed patients. Chromatin structure regulation is important for genome stability maintenance and has the potential to drive genome rearrangements and affect virulence and pathogenesis of pathogens. Here, we performed the first A. fumigatus global chromatin profiling of two histone modifications, H3K4me3 and H3K9me3, focusing on the two most investigated A. fumigatus clinical isolates, Af293 and CEA17. In eukaryotes, H3K4me3 is associated with active transcription, while H3K9me3 often marks silent genes, DNA repeats, and transposons. We found that H3K4me3 deposition is similar between the two isolates, while H3K9me3 is more variable and does not always represent transcriptional silencing. Our work uncovered striking differences in the number, locations, and expression of transposable elements between Af293 and CEA17, and the differences are correlated with H3K9me3 modifications and higher genomic variations among strains of Af293 background. Moreover, we further showed that the Af293 strains from different laboratories actually differ in their genome contents and found a frequently lost region in chromosome VIII. For one such Af293 variant, we identified the chromosomal changes and demonstrated their impacts on its secondary metabolites production, growth and virulence. Overall, our findings not only emphasize the influence of genome heterogeneity on A. fumigatus fitness, but also caution about unnoticed chromosomal variations among common laboratory strains.
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Aspergillus fumigatus/clasificación , Cromosomas Fúngicos/genética , Heterogeneidad Genética , Histonas/metabolismo , Aspergilosis Pulmonar/microbiología , Aspergillus fumigatus/genética , Aspergillus fumigatus/aislamiento & purificación , Cromatina , Elementos Transponibles de ADN , Proteínas Fúngicas/metabolismo , Regulación de la Expresión Génica de las Plantas , Aptitud Genética , Código de Histonas , Humanos , Regiones Promotoras Genéticas , Metabolismo Secundario , VirulenciaRESUMEN
BACKGROUND: Genome-scale metabolic reconstruction tools have been developed in the last decades. They have helped to reconstruct eukaryotic and prokaryotic metabolic models, which have contributed to fields, e.g., genetic engineering, drug discovery, prediction of phenotypes, and other model-driven discoveries. However, the use of these programs requires a high level of bioinformatic skills. Moreover, the functionalities required to build models are scattered throughout multiple tools, requiring knowledge and experience for utilizing several tools. RESULTS: Here we present ChiMera, which combines tools used for model reconstruction, prediction, and visualization. ChiMera uses CarveMe in the reconstruction module, generating a gap-filled draft reconstruction able to produce growth predictions using flux balance analysis for gram-positive and gram-negative bacteria. ChiMera also contains two modules for metabolic network visualization. The first module generates maps for the most important pathways, e.g., glycolysis, nucleotides and amino acids biosynthesis, fatty acid oxidation and biosynthesis and core-metabolism. The second module produces a genome-wide metabolic map, which can be used to retrieve KEGG pathway information for each compound in the model. A module to investigate gene essentiality and knockout is also present. CONCLUSIONS: Overall, ChiMera uses automation algorithms to combine a variety of tools to automatically perform model creation, gap-filling, flux balance analysis (FBA), and metabolic network visualization. ChiMera models readily provide metabolic insights that can aid genetic engineering projects, prediction of phenotypes, and model-driven discoveries.
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Antibacterianos , Bacterias Gramnegativas , Bacterias Grampositivas , Redes y Vías Metabólicas/genética , Genoma BacterianoRESUMEN
Any descriptive language is necessarily metaphoric and interpretative. Two somewhat overlapping-but not identical-languages have been thoroughly employed in the last decade to address the issue of regulatory complexity in biological systems: the terminology of network theory and the jargon of electric circuitry. These approaches have found many formal equivalences between the layout of extant genetic circuits and the architecture of man-made counterparts. However, these languages still fail to describe accurately key features of biological objects, in particular the diversity of signal-transfer molecules and the diffusion that is inherent to any biochemical system. Furthermore, current formalisms associated with networks and circuits can hardly face the problem of multi-scale regulatory complexity-from single molecules to entire ecosystems. We argue that the language of economic theory might be instrumental not only to portray accurately many features of regulatory networks, but also to unveil aspects of the biological complexity problem that remain opaque to other types of analyses. The main perspective opened by the economic metaphor when applied to control of microbiological activities is a focus on metabolism, not gene selfishness, as the necessary background to make sense of regulatory phenomena. As an example, we analyse and reinterpret the widespread phenomenon of catabolite repression with the formal frame of the consumer's choice theory.
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Ecosistema , Metáfora , HumanosRESUMEN
Aspergillus fumigatus is a major cause of human disease. The survival of this fungus is dependent on the cell wall organization and function of its components. The cell wall integrity pathway (CWIP) is the primary signaling cascade that controls de novo synthesis of the cell wall in fungi. Abundant conidiation is a hallmark in A. fumigatus, and uptake of conidia by a susceptible host is usually the initial event in infection. The formation of conidia is mediated by the development of fungus-specific specialized structures, conidiophores, which are accompanied by cell wall remodeling. The molecular regulation of these changes in cell wall composition required for the rise of conidiophore from the solid surface and to disperse the conidia into the air is currently unknown. Here, we investigated the role of CWIP in conidiation. We show that CWIP pkcAG579R, ΔmpkA, and ΔrlmA mutants displayed reduced conidiation during synchronized asexual differentiation. The transcription factor RlmA directly regulated the expression of regulators of conidiation, including flbB, flbC, brlA, abaA, and rasB, as well as genes involved in cell wall synthesis and remodeling, and this affected the chitin content in aerial hyphae. Phosphorylation of RlmA and MpkA was increased during asexual differentiation. We also observed that MpkA physically associated with the proteins FlbB, FlbC, BrlA, and RasB during this process, suggesting another level of cross talk between the CWIP and asexual development pathways. In summary, our results support the conclusion that one function of the CWIP is the regulation of asexual development in filamentous fungi.IMPORTANCE A remarkable feature of the human pathogen Aspergillus fumigatus is its ability to produce impressive amounts of infectious propagules known as conidia. These particles reach immunocompromised patients and may initiate a life-threatening mycosis. The conidiation process in Aspergillus is governed by a sequence of proteins that coordinate the development of conidiophores. This process requires the remodeling of the cell wall so that the conidiophores can rise and withstand the chains of conidia. The events regulating cell wall remodeling during conidiation are currently unknown. Here, we show that the cell wall integrity pathway (CWIP) components RlmA and MpkA directly contribute to the activation of the conidiation cascade by enabling transcription or phosphorylation of critical proteins involved in asexual development. This study points to an essential role for the CWIP during conidiation and provides further insights into the complex regulation of asexual development in filamentous fungi.
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Aspergillus fumigatus/fisiología , Pared Celular/metabolismo , Proteínas Fúngicas/metabolismo , Reproducción Asexuada , Transducción de Señal , Esporas Fúngicas/crecimiento & desarrollo , Aspergilosis/microbiología , Aspergillus fumigatus/crecimiento & desarrollo , Proteínas Fúngicas/genética , HumanosRESUMEN
Metagenomics approaches have been of high relevance for providing enzymes used in diverse industrial applications. In the current study, we have focused on the prospection of protease and glycosyl hydrolase activities from a soil sample by using the lacZα -based plasmid pSEVA232. For this, we used a functional screen based on skimmed milk agar and a pH indicator dye for detection of both enzymes, as previously reported in literature. Although we effectively identified positive clones in the screenings, subsequent experiments revealed that this phenotype was not because of the hydrolytic activity encoded in the metagenomic fragments, but rather due to the insertion of small metagenomic DNA fragments in frame within the coding region of the lacZ gene present in the original vector. Analyses of the thermodynamic stability of mRNA secondary structures indicated that recovering of positive clones was probably due to higher expression levels of the chimeric lacZα-genes in respect to the original from empty vector. We concluded that this method has a higher tendency for recovery false positive clones, when used in combination with a lacZα-based vector. As these vectors are massively used in functional metagenomic screenings, we highlight the importance of reporting boundaries in established metagenomic screenings methodologies.
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Trichoderma reesei is the main filamentous fungus used in industry to produce cellulases. Here we investigated the role of CRZ1 and Ca2+signaling in the fungus T. reesei QM6a concerning holocellulases production. For this, we first searched for potential CRZ1 binding sites in promoter regions of key genes coding holocellulases, as well as transcriptional regulators and sugar and calcium transporters. Using a nearly constructed T. reeseiAcrz1 strain, we demonstrated that most of the genes expected to be regulated by CRZ1 were affected in the mutant strain induced with sugarcane bagasse (SCB) and cellulose. In particular, our data demonstrate that Ca2+ acts synergistically with CRZ1 to modulate gene expression, but also exerts CRZ1-independent regulatory role in gene expression in T. reesei, highlighting the role of the major regulator Ca2+ on the signaling for holocellulases transcriptional control in the most part of cellulases genes here investigated. This work presents new evidence on the regulatory role of CRZ1 and Ca2+ sensing in the regulation of cellulolytic enzymes in T. reesei, evidencing significant and previously unknown function of this Ca2+sensing system in the control key transcriptional regulators (XYR1 and CRE1) and on the expression of genes related to sugar and Ca2+ transport.
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BACKGROUND: Rhodosporidium toruloides is a promising host for the production of bioproducts from lignocellulosic biomass. A key prerequisite for efficient pathway engineering is the availability of robust genetic tools and resources. However, there is a lack of characterized promoters to drive expression of heterologous genes for strain engineering in R. toruloides. RESULTS: This data describes a set of native R. toruloides promoters, characterized over time in four different media commonly used for cultivation of this yeast. The promoter sequences were selected using transcriptional analysis and several of them were found to drive expression bidirectionally. Promoter expression strength was determined by measurement of EGFP and mRuby2 reporters by flow cytometry. A total of 20 constitutive promoters (12 monodirectional and 8 bidirectional) were found, and are expected to be of potential value for genetic engineering of R. toruloides. CONCLUSIONS: A set of robust and constitutive promoters to facilitate genetic engineering of R. toruloides is presented here, ranging from a promoter previously used for this purpose (P7, glyceraldehyde 3-phosphate dehydrogenase, GAPDH) to stronger monodirectional (e.g., P15, mitochondrial adenine nucleotide translocator, ANT) and bidirectional (e.g., P9 and P9R, histones H3 and H4, respectively) promoters. We also identified promoters that may be useful for specific applications such as late-stage expression (e.g., P3, voltage-dependent anion channel protein 2, VDAC2). This set of characterized promoters significantly expands the range of engineering tools available for this yeast and can be applied in future metabolic engineering studies.
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Ingeniería Metabólica , Regiones Promotoras Genéticas , Rhodotorula/genética , Secuencia de Bases , Rhodotorula/metabolismo , Transformación GenéticaRESUMEN
We present a collection of minimalist binary vectors for transformation through ATMT applicable to several fungi species. pLUO plasmid binary vectors consist of a reporter module containing fluorescent proteins, mCherry or eGFP, flanked by a multiple cloning site and a transcription terminator site. They also present a synthetic gene allowing resistance to Hygromicin B flanked by alternate promoters, one for yeast and another for filamentous fungi. Left and right borders were added for Agrobacterium tumefaciens recognition, and a minimal broad-host range RK2 replication origin. Transformation was validated in the pathogenic fungus Paracoccidioides lutzii. Hence, we developed an efficient and reliable molecular tool for fungal transformation: minimalist, synthetic, modular, and available in four different versions, and these can still be readily modified using a few primers and few cloning steps.
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Propionate is an abundant catabolite in nature and represents a rich potential source of carbon for the organisms that can utilize it. However, propionate and propionate-derived catabolites are also toxic to cells, so propionate catabolism can alternatively be viewed as a detoxification mechanism. In this review, we summarize recent progress made in understanding how prokaryotes catabolize propionic acid, how these pathways are regulated and how they might be exploited to develop novel antibacterial interventions.
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Bacterias/metabolismo , Bacterias/patogenicidad , Citratos/metabolismo , Regulación Bacteriana de la Expresión Génica , Propionatos/metabolismo , Bacterias/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Redes y Vías Metabólicas/genética , Operón/genética , Propionatos/toxicidad , VirulenciaRESUMEN
Metal restriction imposed by mammalian hosts during an infection is a common mechanism of defence to reduce or avoid the pathogen infection. Metals are essential for organism survival due to its involvement in several biological processes. Aspergillus fumigatus causes invasive aspergillosis, a disease that typically manifests in immunocompromised patients. A. fumigatus PpzA, the catalytic subunit of protein phosphatase Z (PPZ), has been recently identified as associated with iron assimilation. A. fumigatus has 2 high-affinity mechanisms of iron acquisition during infection: reductive iron assimilation and siderophore-mediated iron uptake. It has been shown that siderophore production is important for A. fumigatus virulence, differently to the reductive iron uptake system. Transcriptomic and proteomic comparisons between ∆ppzA and wild-type strains under iron starvation showed that PpzA has a broad influence on genes involved in secondary metabolism. Liquid chromatography-mass spectrometry under standard and iron starvation conditions confirmed that the ΔppzA mutant had reduced production of pyripyropene A, fumagillin, fumiquinazoline A, triacetyl-fusarinine C, and helvolic acid. The ΔppzA was shown to be avirulent in a neutropenic murine model of invasive pulmonary aspergillosis. PpzA plays an important role at the interface between iron starvation, regulation of SM production, and pathogenicity in A. fumigatus.
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Aspergillus fumigatus/enzimología , Aspergillus fumigatus/patogenicidad , Hierro/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Metabolismo Secundario , Animales , Aspergillus fumigatus/genética , Aspergillus fumigatus/metabolismo , Cromatografía Liquida , Modelos Animales de Enfermedad , Eliminación de Gen , Perfilación de la Expresión Génica , Aspergilosis Pulmonar Invasiva/microbiología , Aspergilosis Pulmonar Invasiva/patología , Espectrometría de Masas , Metabolómica , Ratones , Fosfoproteínas Fosfatasas/genética , Proteoma/análisis , VirulenciaRESUMEN
The serine-threonine kinase TOR, the Target of Rapamycin, is an important regulator of nutrient, energy and stress signaling in eukaryotes. Sch9, a Ser/Thr kinase of AGC family (the cAMP-dependent PKA, cGMP- dependent protein kinase G and phospholipid-dependent protein kinase C family), is a substrate of TOR. Here, we characterized the fungal opportunistic pathogen Aspergillus fumigatus Sch9 homologue (SchA). The schA null mutant was sensitive to rapamycin, high concentrations of calcium, hyperosmotic stress and SchA was involved in iron metabolism. The ΔschA null mutant showed increased phosphorylation of SakA, the A. fumigatus Hog1 homologue. The schA null mutant has increased and decreased trehalose and glycerol accumulation, respectively, suggesting SchA performs different roles for glycerol and trehalose accumulation during osmotic stress. The schA was transcriptionally regulated by osmotic stress and this response was dependent on SakA and MpkC. The double ΔschA ΔsakA and ΔschA ΔmpkC mutants were more sensitive to osmotic stress than the corresponding parental strains. Transcriptomics and proteomics identified direct and indirect targets of SchA post-exposure to hyperosmotic stress. Finally, ΔschA was avirulent in a low dose murine infection model. Our results suggest there is a complex network of interactions amongst the A. fumigatus TOR, SakA and SchA pathways.
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Aspergillus fumigatus/enzimología , Aspergillus fumigatus/patogenicidad , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Animales , Aspergilosis/microbiología , Aspergillus fumigatus/metabolismo , Femenino , Proteínas Fúngicas/metabolismo , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Endogámicos BALB C , Presión Osmótica/fisiología , Estrés Oxidativo/genética , Estrés Oxidativo/fisiología , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Sirolimus/farmacología , Esporas Fúngicas/metabolismo , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , VirulenciaRESUMEN
Pseudomonas putida mt-2 metabolizes m-xylene and other aromatic compounds through the enzymes encoded by the xyl operons of the TOL plasmid pWW0 along with other chromosomally encoded activities. Tiling arrays of densely overlapping oligonucleotides were designed to cover every gene involved in this process, allowing dissection of operon structures and exposing the interplay of plasmid and chromosomal functions. All xyl sequences were transcribed in response to aromatic substrates and the 3'-termini of both upper and lower mRNA operons extended beyond their coding regions, i.e. the 3'-end of the lower operon mRNA penetrated into the convergent xylS regulatory gene. Furthermore, xylR mRNA for the master m-xylene responsive regulator of the system was decreased by aromatic substrates, while the cognate upper operon mRNA was evenly stable throughout its full length. RNA sequencing confirmed these data at a single nucleotide level and refined the formerly misannotated xylL sequence. The chromosomal ortho route for degradation of benzoate (the ben, cat clusters and some pca genes) was activated by this aromatic, but not by the TOL substrates, toluene or m-xylene. We advocate this scenario as a testbed of natural retroactivity between a pre-existing metabolic network and a new biochemical pathway implanted through gene transfer.
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Proteínas Bacterianas/genética , Benzoatos/metabolismo , Proteínas de Unión al ADN/genética , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Tolueno/metabolismo , Factores de Transcripción/genética , Xilenos/metabolismo , Biodegradación Ambiental , Genes Reguladores/genética , Operón/genética , Plásmidos/genética , Pseudomonas putida/enzimología , ARN Mensajero/genéticaRESUMEN
Filamentous fungi are remarkable organisms naturally specialized in deconstructing plant biomass and this feature has a tremendous potential for biofuel production from renewable sources. The past decades have been marked by a remarkable progress in the genetic engineering of fungi to generate industry-compatible strains needed for some biotech applications. In this sense, progress in this field has been marked by the utilization of high-throughput techniques to gain deep understanding of the molecular machinery controlling the physiology of these organisms, starting thus the Systems Biology era of fungi. Additionally, genetic engineering has been extensively applied to modify wellcharacterized promoters in order to construct new expression systems with enhanced performance under the conditions of interest. In this review, we discuss some aspects related to significant progress in the understating and engineering of fungi for biotechnological applications, with special focus on the construction of synthetic promoters and circuits in organisms relevant for industry. Different engineering approaches are shown, and their potential and limitations for the construction of complex synthetic circuits in these organisms are examined. Finally, we discuss the impact of engineered promoter architecture in the single-cell behavior of the system, an often-neglected relationship with a tremendous impact in the final performance of the process of interest. We expect to provide here some new directions to drive future research directed to the construction of high-performance, engineered fungal strains working as microbial cell factories.
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Carbon catabolite repression (CCR) mediated by CRE1 in Trichoderma reesei emerged as a mechanism by which the fungus could adapt to new environments. In the presence of readily available carbon sources such as glucose, the fungus activates this mechanism and inhibits the production of cellulolytic complex enzymes to avoid unnecessary energy expenditure. CCR has been well described for the growth of T. reesei in cellulose and glucose, however, little is known about this process when the carbon source is sophorose, one of the most potent inducers of cellulase production. Thus, we performed high-throughput RNA sequencing to better understand CCR during cellulase formation in the presence of sophorose, by comparing the mutant ∆cre1 with its parental strain, QM9414. Of the 9129 genes present in the genome of T. reesei, 184 were upregulated and 344 downregulated in the mutant strain ∆cre1 compared to QM9414. Genes belonging to the CAZy database, and those encoding transcription factors and transporters are among the gene classes that were repressed by CRE1 in the presence of sophorose; most were possible indirectly regulated by CRE1. We also observed that CRE1 activity is carbon-dependent. A recent study from our group showed that in cellulose, CRE1 repress different groups of genes when compared to sophorose. CCR differences between these carbon sources may be due to the release of cellodextrins in the cellulose polymer, resulting in different targets of CRE1 in both carbon sources. These results contribute to a better understanding of CRE1-mediated CCR in T. reesei when glucose comes from a potent inducer of cellulase production such as sophorose, which could prove useful in improving cellulase production by the biotechnology sector.
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BACKGROUND: Trichoderma reesei is used for industry-scale production of plant cell wall-degrading enzymes, in particular cellulases, but also xylanases. The expression of the encoding genes was so far primarily investigated on the level of transcriptional regulation by regulatory proteins. Otherwise, the impact of chromatin remodelling on gene expression received hardly any attention. In this study we aimed to learn if the chromatin status changes in context to the applied conditions (repressing/inducing), and if the presence or absence of the essential transactivator, the Xylanase regulator 1 (Xyr1), influences the chromatin packaging. RESULTS: Comparing the results of chromatin accessibility real-time PCR analyses and gene expression studies of the two prominent cellulase-encoding genes, cbh1 and cbh2, we found that the chromatin opens during sophorose-mediated induction compared to D-glucose-conferred repression. In the strain bearing a xyr1 deletion the sophorose mediated induction of gene expression is lost and the chromatin opening is strongly reduced. In all conditions the chromatin got denser when Xyr1 is absent. In the case of the xylanase-encoding genes, xyn1 and xyn2, the result was similar concerning the condition-specific response of the chromatin compaction. However, the difference in chromatin status provoked by the absence of Xyr1 is less pronounced. A more detailed investigation of the DNA accessibility in the cbh1 promoter showed that the deletion of xyr1 changed the in vivo footprinting pattern. In particular, we detected increased hypersensitivity on Xyr1-sites and stronger protection of Cre1-sites. Looking for the players directly causing the observed chromatin remodelling, a whole transcriptome shotgun sequencing revealed that 15 genes encoding putative chromatin remodelers are differentially expressed in response to the applied condition and two amongst them are differentially expressed in the absence of Xyr1. CONCLUSIONS: The regulation of xylanase and cellulase expression in T. reesei is not only restricted to the action of transcription factors but is clearly related to changes in the chromatin packaging. Both the applied condition and the presence of Xyr1 influence chromatin status.
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Celulasa/genética , Ensamble y Desensamble de Cromatina/genética , Cromatina/genética , Trichoderma/genética , Celulasas/genética , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica/genética , Glucosa/genética , Regiones Promotoras Genéticas/genética , Transactivadores/genética , Transcripción Genética/genética , Transcriptoma/genéticaRESUMEN
Pseudomonas putidaâ mt-2 encompasses two alternative and potentially conflicting routes for benzoate metabolism, one meta pathway encoded by xyl genes of the pWW0 plasmid and mastered by the Pm promoter and XylS, and one chromosomally encoded ortho pathway initiated by Pben and the BenR protein. Any cross-activation of Pben promoter by XylS ought to cause a metabolic conflict during the degradation of m-xylene because 3-methylbenzoate (3MBz) generated as an intermediate can be channelled through the ortho pathway and produce toxic dead-end metabolites. The activation of Pben by XylS was revisited using both reporter technology and tiling arrays targeted to the sequences of interest around messenger RNA initiation of both Pben and Pm promoters. Analysis of supersensitive luxCDABE fusions, inspection of xylX versus benA transcripts and growth tests of benR mutants indicated that the natural expression ranges of XylS under various conditions are insufficient to cause a significant cross-regulation of Pben whether cells face endogenous or exogenous 3MBz. This seems to stem from the nature of the operators for binding either transcriptional factor, which in the case of the Pben promoter of P. putidaâ mt-2 appear to have evolved for avoiding a strong interaction with XylS.
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Proteínas Bacterianas/metabolismo , Benzoatos/metabolismo , Regulación Bacteriana de la Expresión Génica , Regiones Promotoras Genéticas , Pseudomonas putida/genética , Transactivadores/metabolismo , Xilenos/metabolismo , Proteínas Bacterianas/genética , Pseudomonas putida/crecimiento & desarrollo , Pseudomonas putida/metabolismo , Transactivadores/genética , Activación TranscripcionalRESUMEN
Robustness is the quality of any relational object (biological or otherwise) to maintain its components, its structure, and its function despite both external changes and endogenous fluctuations. Live systems are surprisingly robust, as they are able to not only preserve their physicochemical architecture in the face of variable nutritional and environmental conditions, but also tolerate stochastic variability in the concentrations of their components, fix errors resulting from hazardous events, and make virtually perfect copies of themselves. These qualities have started to be comprehended in full only since the application of network theory formalisms to regulatory phenomena. This review addresses the distinct role of network architecture (topology, logic) and biochemical/kinetic parameters in the materialization of various archetypical robust gene expression circuits in prokaryotes. Some take-home lessons for the construction of artificial regulatory networks (one of the trademarks of synthetic biology) are to be derived from such state of affairs.
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Archaea/fisiología , Fenómenos Fisiológicos Bacterianos , Regulación de la Expresión Génica Arqueal , Regulación Bacteriana de la Expresión Génica , Células Procariotas/fisiología , Adaptación Fisiológica , Estrés FisiológicoRESUMEN
The 'Standard European Vector Architecture' database (SEVA-DB, http://seva.cnb.csic.es) was conceived as a user-friendly, web-based resource and a material clone repository to assist in the choice of optimal plasmid vectors for de-constructing and re-constructing complex prokaryotic phenotypes. The SEVA-DB adopts simple design concepts that facilitate the swapping of functional modules and the extension of genome engineering options to microorganisms beyond typical laboratory strains. Under the SEVA standard, every DNA portion of the plasmid vectors is minimized, edited for flaws in their sequence and/or functionality, and endowed with physical connectivity through three inter-segment insulators that are flanked by fixed, rare restriction sites. Such a scaffold enables the exchangeability of multiple origins of replication and diverse antibiotic selection markers to shape a frame for their further combination with a large variety of cargo modules that can be used for varied end-applications. The core collection of constructs that are available at the SEVA-DB has been produced as a starting point for the further expansion of the formatted vector platform. We argue that adoption of the SEVA format can become a shortcut to fill the phenomenal gap between the existing power of DNA synthesis and the actual engineering of predictable and efficacious bacteria.
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Bases de Datos Genéticas , Vectores Genéticos , Plásmidos/genética , Bacterias/genética , Clonación Molecular , Farmacorresistencia Microbiana/genética , Vectores Genéticos/normas , Internet , Fenotipo , Regiones Promotoras Genéticas , Origen de Réplica , Terminología como AsuntoRESUMEN
Bacteria display considerable cell-to-cell heterogeneity in a number of genetic and physiological traits. Stochastic differences in regulatory patterns (e.g. at the transcriptional level) propagate into the metabolic and physiological status of otherwise isogenic cells, which ultimately results in appearance of sub-populations within the community. As new technologies emerge and because novel single cell strategies are constantly being refined, our knowledge on microbial individuality is in burgeoning and constant expansion. These approaches encompass not only molecular biology tools (e.g. fluorescent-protein based reporters) but also a suite of sophisticated, non-invasive technologies to gain insight into the metabolic state of individual cells. Defining the role of individual heterogeneities is thus instrumental for the population-level understanding of macroscopic processes in both environmental and industrial set-ups. The present article reviews the state-of-the-art methodologies for the investigation of single bacteria at both the genetic and metabolic level, and places the application of currently available tools in the context of microbial ecology and environmental microbiology. As a case example, we examine the stochastic and multi-stable behaviour of the TOL-encoded pathway of Pseudomonas putida mt-2 for the biodegradation of aromatic compounds. Bet-hedging strategies and division of labour are considered as factors pushing forward the evolution of environmental microorganisms.
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Microbiología Ambiental , Contaminantes Ambientales/metabolismo , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Biodegradación Ambiental , Variación Genética , Genotipo , Fenotipo , Plásmidos/genéticaRESUMEN
The ascomycete Trichoderma reesei is one of the most well-studied cellulolytic fungi and is widely used by the biotechnology industry in the production of second generation bioethanol. The carbon catabolite repression (CCR) mechanism adopted by T. reesei is mediated by the transcription factor CRE1. CCR represses genes related to cellulase production when a carbon source is readily available in the medium. Using RNA sequencing, we investigated CCR during the synthesis of cellulases, comparing the T. reesei Δcre1 mutant strain with its parental strain, QM9414. Of 9129 genes in the T. reesei genome, 268 genes were upregulated and 85 were downregulated in the presence of cellulose (Avicel). In addition, 251 genes were upregulated and 230 were downregulated in the presence of a high concentration of glucose. Genes encoding cellulolytic enzymes and transcription factors and genes related to the transport of nutrients and oxidative metabolism were also targets of CCR, mediated by CRE1 in a carbon source-dependent manner. Our results also suggested that CRE1 regulates the expression of genes related to the use of copper and iron as final electron acceptors or as cofactors of enzymes that participate in biomass degradation. As a result, the final effect of CRE1-mediated transcriptional regulation is to modulate the access of cellulolytic enzymes to cellulose polymers or blocks the entry of cellulase inducers into the cell, depending on the glucose content in the medium. These results will contribute to a better understanding of the mechanism of carbon catabolite repression in T. reesei, thereby enhancing its application in several biotechnology fields.