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

P-Ser-HPr--a link between carbon metabolism and the virulence of some pathogenic bacteria

International audienceHPr kinase/phosphorylase phosphorylates HPr, a phosphocarrier protein of the phosphoenolpyruvate:carbohydrate phosphotransferase system, at serine-46. P-Ser-HPr is the central regulator of carbon metabolism in Gram-positive bacteria, but also plays a role in virulence development of certain pathogens. In Listeria monocytogenes, several virulence genes, which depend on the transcription activator PrfA, are repressed by glucose, fructose, etc., in a catabolite repressor (CcpA)-independent mechanism. However, the catabolite co-repressor P-Ser-HPr was found to inhibit the activity of PrfA. In an hprKV267F mutant, in which most of the HPr is transformed into P-Ser-HPr, PrfA was barely active. The ptsH1 mutation (Ser-46 of HPr replaced with an alanine) prevented the inhibitory effect of the hprKV267F mutation. Interestingly, disruption of ccpA also inhibited PrfA activity. This effect is probably also mediated via P-Ser-HPr, since ccpA disruption leads to elevated amounts of P-Ser-HPr. Indeed, a ccpA ptsH1 double mutant exhibited normal PrfA activity. In S. pyogenes, the expression of several virulence genes depends on the transcription activator Mga. Interestingly, the mga promoter is preceded by an operator site, which serves as target for the CcpA/P-Ser-HPr complex. Numerous Gram-negative pathogens also contain hprK, which is often organised in an operon with transcription regulators necessary for the development of virulence, indicating that in these organisms P-Ser-HPr also plays a role in pathogenesis. Indeed, inactivation of Neisseria meningitidis hprK strongly diminished cell adhesion of this pathogen

Development of an in silico method for the identification of subcomplexes involved in the biogenesis of multiprotein complexes in Saccharomyces cerevisiae

Abstract Background Large sets of protein-protein interaction data coming either from biological experiments or predictive methods are available and can be combined to construct networks from which information about various cell processes can be extracted. We have developed an in silico approach based on these information to model the biogenesis of multiprotein complexes in the yeast Saccharomyces cerevisiae. Results Firstly, we have built three protein interaction networks by collecting the protein-protein interactions, which involved the subunits of three complexes, from different databases. The protein-protein interactions come from different kinds of biological experiments or are predicted. We have chosen the elongator and the mediator head complexes that are soluble and exhibit an architecture with subcomplexes that could be functional modules, and the mitochondrial bc 1 complex, which is an integral membrane complex and for which a late assembly subcomplex has been described. Secondly, by applying a clustering strategy to these networks, we were able to identify subcomplexes involved in the biogenesis of the complexes as well as the proteins interacting with each subcomplex. Thirdly, in order to validate our in silico results for the cytochrome bc1 complex we have analysed the physical interactions existing between three subunits by performing immunoprecipitation experiments in several genetic context. Conclusions For the two soluble complexes (the elongator and mediator head), our model shows a strong clustering of subunits that belong to a known subcomplex or module. For the membrane bc 1 complex, our approach has suggested new interactions between subunits in the early steps of the assembly pathway that were experimentally confirmed. Scripts can be downloaded from the site: http://bim.igmors.u-psud.fr/isips

Ribosome recycling defects modify the balance between the synthesis and assembly of specific subunits of the oxidative phosphorylation complexes in yeast mitochondria

Mitochondria have their own translation machinery that produces key subunits of the OXPHOS complexes. This machinery relies on the coordinated action of nuclear-encoded factors of bacterial origin that are well conserved between humans and yeast. In humans, mutations in these factors can cause diseases; in yeast, mutations abolishing mitochondrial translation destabilize the mitochondrial DNA. We show that when the mitochondrial genome contains no introns, the loss of the yeast factors Mif3 and Rrf1 involved in ribosome recycling neither blocks translation nor destabilizes mitochondrial DNA. Rather, the absence of these factors increases the synthesis of the mitochondrially-encoded subunits Cox1, Cytb and Atp9, while strongly impairing the assembly of OXPHOS complexes IV and V. We further show that in the absence of Rrf1, the COX1 specific translation activator Mss51 accumulates in low molecular weight forms, thought to be the source of the translationally-active form, explaining the increased synthesis of Cox1. We propose that Rrf1 takes part in the coordination between translation and OXPHOS assembly in yeast mitochondria. These interactions between general and specific translation factors might reveal an evolutionary adaptation of the bacterial translation machinery to the set of integral membrane proteins that are translated within mitochondria

Additional file 1: Figure S1. of Development of an in silico method for the identification of subcomplexes involved in the biogenesis of multiprotein complexes in Saccharomyces cerevisiae

Assembly models obtained with different similarity scores. Panel A: hierarchical tree representing the distances between the seven subunits of the Mediator Head complex. Upper Part: tree obtained with the Dice similarity score. Lower part: tree obtained with the pseudo-Jaccard similarity score. Panel B: hierarchical trees representing the distances between the ten subunits of the bc1 complex. Upper part: tree obtained with the Dice similarity score. Lower part: tree obtained with the MS or pseudo-Jaccard similarity score. (PDF 107 kb

Chemicals or mutations that target mitochondrial translation can rescue the respiratory deficiency of yeast bcs1 mutants

Bcs1p is a chaperone that is required for the incorporation of the Rieske subunit within complex III of the mitochondrial respiratory chain. Mutations in the human gene BCS1L (BCS1-like) are the most frequent nuclear mutations resulting in complex III-related pathologies. In yeast, the mimicking of some pathogenic mutations causes a respiratory deficiency. We have screened chemical libraries and found that two antibiotics, pentamidine and clarithromycin, can compensate two bcs1 point mutations in yeast, one of which is the equivalent of a mutation found in a human patient. As both antibiotics target the large mtrRNA of the mitoribosome, we focused our analysis on mitochondrial translation. We found that the absence of non-essential translation factors Rrf1 or Mif3, which act at the recycling/initiation steps, also compensates for the respiratory deficiency of yeast bcs1 mutations. At compensating concentrations, both antibiotics, as well as the absence of Rrf1, cause an imbalanced synthesis of respiratory subunits which impairs the assembly of the respiratory complexes and especially that of complex IV. Finally, we show that pentamidine also decreases the assembly of complex I in nematode mitochondria. It is well known that complexes III and IV exist within the mitochondrial inner membrane as supramolecular complexes III2/IV in yeast or I/III2/IV in higher eukaryotes. Therefore, we propose that the changes in mitochondrial translation caused by the drugs or by the absence of translation factors, can compensate for bcs1 mutations by modifying the equilibrium between illegitimate, and thus inactive, and active supercomplexes

Enterococcus faecalis utilizes maltose by connecting two incompatible metabolic routes via a novel maltose 6-phosphate 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--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 phospho-substrates revealed that MapP preferably dephosphorylates disaccharides containing an O--glycosyl linkage

Artemisinin and its derivatives target mitochondrial c-type cytochromes in yeast and human cells

[email protected]@i2bc.paris-saclay.frInternational audienceArtemisinin and its derivatives kill malaria parasites and inhibit the proliferation of cancer cells. In both processes, heme was shown to play a key role in artemisinin bioactivation. We found that artemisinin and clinical artemisinin derivatives are able to compensate for a mutation in the yeast Bcs1 protein, a key chaperon involved in biogenesis of the mitochondrial respiratory complex III. The equivalent Bcs1 variant causes an encephalopathy in human by affecting complex III assembly. We show that artemisinin derivatives decrease the content of mitochondrial cytochromes and disturb the maturation of the complex III cytochrome c1. This last effect is likely responsible for the compensation by decreasing the detrimental over-accumulation of the inactive pre-complex III observed in the bcs1 mutant. We further show that a fluorescent dihydroartemisinin probe rapidly accumulates in the mitochondrial network and targets cytochromes c and c1 in yeast, human cells and isolated mitochondria. In vitro this probe interacts with purified cytochrome c only under reducing conditions and we detected cytochrome c-dihydroartemisinin covalent adducts by mass spectrometry analyses. We propose that reduced mitochondrial c-type cytochromes act as both targets and mediators of artemisinin bioactivation in yeast and human cells

Structural studies of cyclodipeptide synthases with RNA microhelices mimicking their natural tRNA susbtrates

International audienc