Régulation de MtlR, activateur transcriptionnel de l'opéron mtl de Bacillus subtilis, par le domaine EIIB du transporteur du mannitol.

Chez Bacillus subtilis l expression de l opéron mtl pour l utilisation du mannitol est contrôlé par MtlR. MtlR est un activateur transcriptionnel qui appartient à la famille des régulateurs DeoR composé d un domaine HTH suivi de deux PRDs, un domaine EIIBGat et un domaine EIIAMtl-like.Le mécanisme général de la régulation de l activité de MtlR est basé sur sa phosphorylation par des composants du PTS. La phosphorylation sur la Cystéine 419 du domaine EIIBGat par P~EIIAMtl a un effet négatif majeur sur l activité de MtlR. Par conséquent, dans un mutant mtlF où EIIAMtl est délétée MtlR est constitutivement actif.Dans cette étude nous avons mis en évidence un nouveau phénomène de régulation de MtlR impliquant la protéine du PTS, EIIBMtl.Nous avons observé que lorsque on déléte l opéron mtl ou EIIBMtl et EIIAMtl, l activité constitutive de MtlR dans un mutant mtlF déjà observée est abolie d où notre hypothèse que EIIBMtl aura un effet sur l activité de MtlR. Par des expériences de double hybride nous avons montré une interaction directe, spécifique et bidirectionnelle entre les deux protéines EIIBMtl et EIIBGatEIIAMtl-like de MtlR. D une manière comparable à la cellule où EIIBMtl est fusionnée à la protéine EIICMtl nous avons démontré que seulement la forme EIIBMtl fusionné à la perméase EIICMtl est capable d activer MtlR mais nous avons également démontré que ce n est pas EIICMtl qui est essentielle à l interaction entre EIIBMtl et MtlR mais c est le voisinage de la membrane qui est essentielle pour l établissement de cette interaction et l activation de MtlRUn modèle de régulation de l activité du régulateur MtlR est proposé. Dans ce modèle l induction de l opéron mtl via l activation de MtlR requiert la phosphorylation de PRDII de MtlR par P~His-HPr, la déphosphorylation de EIIBGat de MtlR par EIIAMtl et la présence de la forme non-phosphorylée de l EIIBMtl qui est dominante en présence du substrat inducteur, le mannitol. Ainsi, l EIIBMtl non-phosphorylée séquestre MtlR déphosphorylé sur sa cystéine 419 à la membrane, l active et induit l expression de l opéron mtl.The Bacillus subtilis mtl operon encodes the enzymes necessary for mannitol utilization. Its expression is controlled by MtlR, a transcriptional activator belonging to the DeoR family. MtlR contains a HTH domain followed by two PTS regulation domains (PRDs), an EIIBGat domain and an EIIAMtl-like domain.The general mechanism of the regulation of MtlR activity is based on its phosphorylation by components of the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS). The phosphorylation of EIIBGat on cysteine 419 by P~EIIAMtl has a major negative effect on the activity of MtlR. The absence of EIIAMtl in a mtlF mutant therefore leads to constitutively active MtlR.In this study a new mechanism of MtlR regulation based on the interaction of the PTS component EIIBMtl with MtlR is presented.We observed that the deletion of the entire mtlAFD operon or of mtlF and only the 3 -part of mtlA (encoding the EIIBMtl domain) abolishes the constitutive MtlR activity of the mtlF mutant, suggesting that MtlR activity depends on functional EIIBMtl. By carrying out yeast two-hybrid experiments we could establish a direct, specific and bidirectional interaction between EIIBMtl and the EIIBGatEIIAMtl-like part of MtlR.Complementation of the above mutants was possible with entire MtlA, but not with the EIIBMtl domain. EIIBMtl is normally fused to the membrane protein EIICMtl; we therefore fused EIIBMtl to another membrane, which indeed restored MtlR function in the absence of EIICMtl. The EIICMtl domain is therefore not essential for the interaction between EIIBMtl and MtlR; it is rather the vicinity of the membrane which is required for the activation of MtlR.A regulation model of MtlR activity is proposed. In this model, the MtlR-mediated induction of the mtlAFD operon requires the phosphorylation of PRDII by P~His-HPr and the dephosphorylation of EIIBGat by EIIAMtl. The presence of unphosphorylated EIIBMtl, which prevails when the inducer mannitol is present, is also required. Under these conditions unphosphorylated EIIBMtl sequesters MtlR dephosphoryled on cysteine 419, but phosphorylated at His-342, to the membrane thereby activating the transcription activator, which leads to increased expression of the mtlAFD operon.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

CcpA represses the expression of the divergent cit operons of Enterococcus faecalis through multiple cre sites

<p>Abstract</p> <p>Background</p> <p>In <it>Enterococcus faecalis </it>the genes encoding the enzymes involved in citrate metabolism are organized in two divergent operons, <it>citHO </it>and <it>oadHDB-citCDEFX-oadA-citMG </it>(<it>citCL </it>locus). Expression of both operons is specifically activated by adding citrate to the medium. This activation is mediated by binding of the GntR-like transcriptional regulator (CitO) to the <it>cis</it>-acting sequences located in the <it>cit </it>intergenic region. Early studies indicated that citrate and glucose could not be co-metabolized suggesting some form of catabolite repression, however the molecular mechanism remained unknown.</p> <p>Results</p> <p>In this study, we observed that the <it>citHO </it>promoter is repressed in the presence of sugars transported by the Phosphoenolpyruvate:carbohydrate Phosphotranserase System (PTS sugars). This result strongly suggested that Carbon Catabolic Repression (CCR) impedes the expression of the activator CitO and the subsequent induction of the <it>cit </it>pathway. In fact, we demonstrate that CCR is acting on both promoters. It is partially relieved in a <it>ccpA</it>-deficient <it>E. faecalis </it>strain indicating that a CcpA-independent mechanism is also involved in regulation of the two operons. Furthermore, sequence analysis of the <it>citH</it>/<it>oadH </it>intergenic region revealed the presence of three putative catabolite responsive elements (<it>cre</it>). We found that they are all active and able to bind the CcpA/P-Ser-HPr complex, which downregulates the expression of the <it>cit </it>operons. Systematic mutation of the CcpA/P-Ser-HPr binding sites revealed that <it>cre1 </it>and <it>cre2 </it>contribute to <it>citHO </it>repression, while <it>cre3 </it>is involved in CCR of <it>citCL</it></p> <p>Conclusion</p> <p>In conclusion, our study establishes that expression of the <it>cit </it>operons in <it>E. faecalis </it>is controlled by CCR via CcpA-dependent and -independent mechanisms.</p

Ecohydrological consequences of tree removal in an urban park evaluated using open data, free software and a minimalist measuring campaign

With ongoing global climate change and an increasingly urbanized population, the importance of city parks and other forms of urban vegetation increases. Trees in urban parks can play an important role in mitigating runoff and delivering other ecosystem services. Park managers, E-NGOs, citizen scientists and others are increasingly called upon to evaluate the possible consequences of changes in park management such as, e.g., tree removal. Here, we present an unorthodox approach to hydrological modelling and its potential use in local policy making regarding urban greenery. The approach consists of a minimalist field campaign to characterize vegetation and soil moisture status combined with a novel model calibration using freely available data and software. During modelling, we were able to obtain coefficients of determination (R2) of 0.66 and 0.73 for probe-measured and simulated soil moisture under tree stand and park lawn land covers respectively. The results demonstrated that tree cover had a significant positive effect on the hydrological regime of the locality through interception, transpiration and effects on soil moisture. Simulations suggested that tree cover was twice as effective at mitigating runoff than park lawn and almost seven times better than impervious surfaces. In the case of a potential replacement of tree vegetation in favour of park lawn or impervious surfaces an increase in runoff of 14% and 81% respectively could be expected. The main conclusion drawn from our study was that such an approach can be a very useful tool for supporting local decision-making processes as it offers a freely available, cheap and relatively easy-to-use way to describe the hydrological consequences of landcover change (e.g., tree removal) with sufficient accuracy.OA-hybri

Enterococcus faecalis MalR acts as a repressor of the maltose operons and additionally mediates their catabolite repression via direct interaction with seryl-phosphorylated-HPr

Enterococci are gram-positive pathogens and lead to cause hospital-acquired infections worldwide. Central carbon metabolism was shown as highly induced in Enterococcus faecalis during infection context. Metabolism of α-polysaccharides was previously described as an important factor for host colonisation and biofilm formation. A better characterisation of the adaptation of this bacterium to carbohydrate availabilities may lead to a better understanding of the link between carbohydrate metabolism and the infection process of E. faecalis. Here we show that MalR, a LacI/GalR transcriptional regulator, is the main factor in the regulation of the two divergent operons involved in maltose metabolism in this bacterium. The malR gene is transcribed from the malP promoter, but also from an internal promoter inside the gene located upstream of malR. In the absence of maltose, MalR acts as a repressor and in the presence of glucose, it exerts efficient CcpA-independent carbon catabolite repression. The central PTS protein P-Ser-HPr interacts directly with MalR and enhances its DNA binding capacity, which allows E. faecalis to adapt its metabolism to environmental conditions.Fil: Grand, Maxime. Universite de Caen Basse Normandie; FranciaFil: Blancato, Victor Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Espariz, Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Deutscher, Josef. Université Paris-Saclay; Francia. Universite de Paris; FranciaFil: Pikis, Andreas. Center For Drug Evaluation And Research; Estados UnidosFil: Hartke, Axel. Universite de Caen Basse Normandie; FranciaFil: Magni, Christian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Sauvageot, Nicolas. Universite de Caen Basse Normandie; Franci

Functional Characterization of the Incomplete Phosphotransferase System (PTS) of the Intracellular Pathogen Brucella melitensis

Background: In many bacteria, the phosphotransferase system (PTS) is a key player in the regulation of the assimilation of alternative carbon sources notably through catabolic repression. The intracellular pathogens Brucella spp. possess four PTS proteins (EI Ntr, NPr, EIIA Ntr and an EIIA of the mannose family) but no PTS permease suggesting that this PTS might serve only regulatory functions

Ecohydrological consequences of tree removal in an urban park evaluated using open data, free software and a minimalist measuring campaign

With ongoing global climate change and an increasingly urbanized population, the importance of city parks and other forms of urban vegetation increases. Trees in urban parks can play an important role in mitigating runoff and delivering other ecosystem services. Park managers, E-NGOs, citizen scientists and others are increasingly called upon to evaluate the possible consequences of changes in park management such as, e.g., tree removal. Here, we present an unorthodox approach to hydrological modelling and its potential use in local policy making regarding urban greenery. The approach consists of a minimalist field campaign to characterize vegetation and soil moisture status combined with a novel model calibration using freely available data and software. During modelling, we were able to obtain coefficients of determination (R-2) of 0.66 and 0.73 for probe-measured and simulated soil moisture under tree stand and park lawn land covers respectively. The results demonstrated that tree cover had a significant positive effect on the hydrological regime of the locality through interception, transpiration and effects on soil moisture. Simulations suggested that tree cover was twice as effective at mitigating runoff than park lawn and almost seven times better than impervious surfaces. In the case of a potential replacement of tree vegetation in favour of park lawn or impervious surfaces an increase in runoff of 14% and 81% respectively could be expected. The main conclusion drawn from our study was that such an approach can be a very useful tool for supporting local decision-making processes as it offers a freely available, cheap and relatively easy-to-use way to describe the hydrological consequences of landcover change (e.g., tree removal) with sufficient accuracy. (C) 2018 The Authors. Published by Elsevier B.V

Enterococcus faecalis utilizes maltose by connecting two incompatible metabolic routes via a novel maltose-6-P phosphatase (MapP)

Similar to Bacillus subtilis, Enterococcus faecalis transports and phosphorylates maltose via a phosphoenolpyruvate (PEP):maltose phosphotransferase system (PTS). The maltose-specific PTS permease is encoded by the malT gene. However, E. faecalis lacks a malA gene encoding a 6-phospho-a-glucosidase, which in B. subtilis hydrolyses maltose 6-P into glucose and glucose 6-P. Instead, an operon encoding a maltose phosphorylase (MalP), a phosphoglucomutase and a mutarotase starts upstream from malT. MalP was suggested to split maltose 6-P into glucose 1-P and glucose 6-P. However, purified MalP phosphorolyses maltose but not maltose 6-P. We discovered that the gene downstream from malT encodes a novel enzyme (MapP) that dephosphorylates maltose 6-P formed by the PTS. The resulting intracellular maltose is cleaved by MalP into glucose and glucose 1-P. Slow uptake of maltose probably via a maltodextrin ABC transporter allows poor growth for the mapP but not the malP mutant. Synthesis of MapP in a B. subtilis mutant accumulating maltose 6-P restored growth on maltose. MapP catalyses the dephosphorylation of intracellular maltose 6-P, and the resulting maltose is converted by the B. subtilis maltose phosphorylase into glucose and glucose 1-P. MapP therefore connects PTS-mediated maltose uptake to maltose phosphorylase-catalysed metabolism. Dephosphorylation assays with a wide variety of phosphosubstrates revealed that MapP preferably dephosphorylates disaccharides containing an O-aglycosyl linkageFil: Mokhtari, Abdelhamid. Institut National de la Recherche Agronomique. Microbiologie de l’Alimentation au Service de la Santé Humaine; Francia. University Mentouri. Faculty of Natural Science and Life. Department of Biochemistry-Microbiology. Laboratory of Environmental Biology; ArgeliaFil: Blancato, Victor Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Repizo, Guillermo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Henry, Céline. Institut National de la Recherche Agronomique. Microbiologie de l’Alimentation au Service de la Santé Humaine; FranciaFil: Pikis, Andreas. Center for Drug Evaluation and Research. Food and Drug Administration; Estados UnidosFil: Bourand, Alexa. Institut National de la Recherche Agronomique. Microbiologie de l’Alimentation au Service de la Santé Humaine; FranciaFil: Alvarez, Maria de Fatima. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Immel, Stefan. Technische Universität Darmstad. Institut für Organische Chemie; AlemaniaFil: Mechakra Maza, Aicha. University Mentouri. Faculty of Natural Science and Life. Department of Biochemistry-Microbiology. Laboratory of Environmental Biology; ArgeliaFil: Hartke, Axel. Universite de Caen Basse Normandie; FranciaFil: Thompson, John. National Institutes of Health. Laboratory of Cell and Developmental Biology. Microbial Biochemistry and Genetics Section; Estados UnidosFil: Magni, Christian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Deutscher, Josef. Institut National de la Recherche Agronomique. Microbiologie de l’Alimentation au Service de la Santé Humaine; Franci

Structural Basis for the Regulation Mechanism of the Tyrosine Kinase CapB from Staphylococcus aureus

Bacteria were thought to be devoid of tyrosine-phosphorylating enzymes. However, several tyrosine kinases without similarity to their eukaryotic counterparts have recently been identified in bacteria. They are involved in many physiological processes, but their accurate functions remain poorly understood due to slow progress in their structural characterization. They have been best characterized as copolymerases involved in the synthesis and export of extracellular polysaccharides. These compounds play critical roles in the virulence of pathogenic bacteria, and bacterial tyrosine kinases can thus be considered as potential therapeutic targets. Here, we present the crystal structures of the phosphorylated and unphosphorylated states of the tyrosine kinase CapB from the human pathogen Staphylococcus aureus together with the activator domain of its cognate transmembrane modulator CapA. This first high-resolution structure of a bacterial tyrosine kinase reveals a 230-kDa ring-shaped octamer that dissociates upon intermolecular autophosphorylation. These observations provide a molecular basis for the regulation mechanism of the bacterial tyrosine kinases and give insights into their copolymerase function

A Functional Genomics Approach to Establish the Complement of Carbohydrate Transporters in Streptococcus pneumoniae

The aerotolerant anaerobe Streptococcus pneumoniae is part of the normal nasopharyngeal microbiota of humans and one of the most important invasive pathogens. A genomic survey allowed establishing the occurrence of twenty-one phosphotransferase systems, seven carbohydrate uptake ABC transporters, one sodium∶solute symporter and a permease, underlining an exceptionally high capacity for uptake of carbohydrate substrates. Despite high genomic variability, combined phenotypic and genomic analysis of twenty sequenced strains did assign the substrate specificity only to two uptake systems. Systematic analysis of mutants for most carbohydrate transporters enabled us to assign a phenotype and substrate specificity to twenty-three transport systems. For five putative transporters for galactose, pentoses, ribonucleosides and sulphated glycans activity was inferred, but not experimentally confirmed and only one transport system remains with an unknown substrate and lack of any functional annotation. Using a metabolic approach, 80% of the thirty-two fermentable carbon substrates were assigned to the corresponding transporter. The complexity and robustness of sugar uptake is underlined by the finding that many transporters have multiple substrates, and many sugars are transported by more than one system. The present work permits to draw a functional map of the complete arsenal of carbohydrate utilisation proteins of pneumococci, allows re-annotation of genomic data and might serve as a reference for related species. These data provide tools for specific investigation of the roles of the different carbon substrates on pneumococcal physiology in the host during carriage and invasive infection

The Thermoanaerobacter Glycobiome Reveals Mechanisms of Pentose and Hexose Co-Utilization in Bacteria

Author Summary Renewable liquid fuels derived from lignocellulosic biomass could alleviate global energy shortage and climate change. Cellulose and hemicellulose are the main components of lignocellulosic biomass. Therefore, the ability to simultaneously utilize pentose and hexose (i.e., co-utilization) has been a crucial challenge for industrial microbes producing lignocellulosic biofuels. Certain thermoanaerobic bacteria demonstrate this unusual talent, but the genetic foundation and molecular mechanism of this process remain unknown. In this study, we reconstructed the structure and dynamics of the first genome-wide carbon utilization network of thermoanaerobes. This transcriptome-based co-expression network reveals that glucose, xylose, fructose, and cellobiose catabolism are each featured on distinct functional modules. Furthermore, the dynamics of the network suggests a distinct yet collaborative nature between glucose and xylose catabolism. In addition, we experimentally demonstrated that these novel network-derived features can be rationally exploited for product-yield enhancement via optimized timing and balanced loading of the carbon supply in a substrate-specific manner. Thus, the newly discovered modular and precisely regulated network elucidates unique features of thermoanaerobic glycobiomes and reveals novel perturbation strategies and targets for the enhanced thermophilic production of lignocellulosic biofuels.Yeshttp://www.plosgenetics.org/static/editorial#pee