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Accueil > Départements > Microbiologie > Marie-Joëlle VIROLLE : Métabolisme Energétique des Streptomyces

Publications de l’équipe

2019


  • S. Werten, N. H. Rustmeier, M. Gemmer, M. - J. Virolle, et W. Hinrichs, « Structural and Biochemical Analysis of a Phosin from Streptomyces chartreusis Reveals a Combined Polyphosphate- and Metal-Binding Fold », FEBS letters, vol. 593, nᵒ 15, p. 2019-2029, août 2019.
    Résumé : X-ray crystallographic analysis of a phosin (PptA) from Steptomyces chartreusis reveals a metal-associated, lozenge-shaped fold featuring a 5-10 Å wide, positively charged tunnel that traverses the protein core. Two distinct metal-binding sites were identified in which the predominant metal ion was Cu2+ . In solution, PptA forms stable homodimers that bind with nanomolar affinity to polyphosphate, a stress-related biopolymer acting as a phosphate and energy reserve in conditions of nutrient depletion. A single protein dimer interacts with 14-15 consecutive phosphate moieties within the polymer. Our observations suggest that PptA plays a role in polyphosphate metabolism, mobilisation or sensing, possibly by acting in concert with polyphosphate kinase (Ppk). Like Ppk, phosins may inuence antibiotic synthesis by streptomycetes. This article is protected by copyright. All rights reserved.
    Mots-clés : antibiotics, Conserved histidine α-helical domain (CHAD), MESMIC, MICROBIO, nutritional stress, phosphate metabolism, secondary metabolism, signalling.

2017



  • C. Esnault, T. Dulermo, A. Smirnov, A. Askora, M. David, A. Deniset-Besseau, I. - B. Holland, et M. - J. Virolle, « Strong antibiotic production is correlated with highly active oxidative metabolism in Streptomyces coelicolor M145 », Scientific Reports, vol. 7, nᵒ 1, 2017.

  • C. Esnault, D. Leiber, C. Toffano-Nioche, Z. Tanfin, et M. - J. Virolle, « Another example of enzymatic promiscuity: the polyphosphate kinase of Streptomyces lividans is endowed with phospholipase D activity », Applied Microbiology and Biotechnology, vol. 101, nᵒ 1, p. 139-145, janv. 2017.
    Résumé : Polyphosphate kinases (PPK) from different bacteria, including that of Streptomyces lividans, were shown to contain the typical HKD motif present in phospholipase D (PLD) and showed structural similarities to the latter. This observation prompted us to investigate the PLD activity of PPK of S. lividans, in vitro. The ability of PPK to catalyze the hydrolysis of phosphatidylcholine (PC), the PLD substrate, was assessed by the quantification of [(3)H]phosphatidic acid (PA) released from [(3)H]PC-labeled ELT3 cell membranes. Basal cell membrane PLD activity as well as GTPγS-activated PLD activity was higher in the presence than in absence of PPK. After abolition of the basal PLD activity of the membranes by heat or tryptic treatment, the addition of PPK to cell membranes was still accompanied by an increased production of PA demonstrating that PPK also bears a PLD activity. PLD activity of PPK was also assessed by the production of choline from hydrolysis of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) in the presence of the Amplex Red reagent and compared to two commercial PLD enzymes. These data demonstrated that PPK is endowed with a weak but clearly detectable PLD activity. The question of the biological signification, if any, of this enzymatic promiscuity is discussed.
    Mots-clés : Amino Acid Motifs, Cell Membrane, Choline, DBG, eBio, Hydrolysis, Lipid droplets, MESMIC, MICROBIO, PF, Phosphatidic Acids, Phosphatidylcholines, Phospholipase D, Phosphotransferases (Phosphate Group Acceptor), Polyphosphate kinase, Promiscuous enzyme, Protein Conformation, SSFA, Streptomyces lividans.

  • A. Millan-Oropeza, C. Henry, M. Blein-Nicolas, A. Aubert-Frambourg, F. Moussa, J. Bleton, et M. - J. Virolle, « Quantitative Proteomics Analysis Confirmed Oxidative Metabolism Predominates in Streptomyces coelicolor versus Glycolytic Metabolism in Streptomyces lividans », Journal of Proteome Research, juin 2017.
    Résumé : Recent physiological studies indicated that S. lividans metabolism was mainly glycolytic, whereas S. coelicolor metabolism was mainly oxidative. To determine whether such metabolic characteristics were correlated with consistent proteomics features, a comparative label-free, shotgun proteomics analysis of these strains was carried out. Among 2024 proteins identified, 360 showed significant differences in abundance between the strains. This study revealed that S. coelicolor catabolized glucose less actively than S. lividans, whereas the amino acids present in the medium were catabolized less actively by S. lividans than by S. coelicolor. The abundance of glycolytic proteins in S. lividans was consistent with its high glycolytic activity, whereas the abundance of proteins involved in the catabolism of amino acids in S. coelicolor provided an explanatory basis for its predominantly oxidative metabolism. In this study, conducted under conditions of low O2 availability, proteins involved in resistance to oxidative stress and those belonging to a DosR-like dormancy regulon were abundant in S. coelicolor, whereas tellurium resistance proteins were abundant in S. lividans. This indicated that the strains reacted differently to O2 limitation. Proteins belonging to the CDA, RED, and ACT pathways, usually highly expressed in S. coelicolor, were not detected under these conditions, whereas proteins of siderophores, 5-hydroxyectoine, and terpenoid biosynthetic pathways were present.
    Mots-clés : dormancy regulon, glycolytic metabolism, Mass Spectrometry, MESMIC, MICROBIO, oxidative metabolism, oxidative stress, Secondary metabolites, shotgun label-free quantitative proteomics, Streptomyces, tellurium resistance proteins.

  • A. Millan-Oropeza, R. Rebois, M. David, F. Moussa, A. Dazzi, J. Bleton, M. - J. Virolle, et A. Deniset-Besseau, « Attenuated Total Reflection Fourier Transform Infrared (ATR FT-IR) for Rapid Determination of Microbial Cell Lipid Content: Correlation with Gas Chromatography-Mass Spectrometry (GC-MS) », Applied Spectroscopy, p. 3702817709459, janv. 2017.
    Résumé : There is a growing interest worldwide for the production of renewable oil without mobilizing agriculture lands; fast and reliable methods are needed to identify highly oleaginous microorganisms of potential industrial interest. The aim of this study was to demonstrate the relevance of attenuated total reflection (ATR) spectroscopy to achieve this goal. To do so, the total lipid content of lyophilized samples of five Streptomyces strains with varying lipid content was assessed with two classical quantitative but time-consuming methods, gas chromatography-mass spectrometry (GC-MS) and ATR Fourier transform infrared (ATR FT-IR) spectroscopy in transmission mode with KBr pellets and the fast ATR method, often questioned for its lack of reliability. A linear correlation between these three methods was demonstrated allowing the establishment of equations to convert ATR values expressed as CO/amide I ratio, into micrograms of lipid per milligram of biomass. The ATR method proved to be as reliable and quantitative as the classical GC-MS and FT-IR in transmission mode methods but faster and more reproducible than the latter since it involves far less manipulation for sample preparation than the two others. Attenuated total reflection could be regarded as an efficient fast screening method to identify natural or genetically modified oleaginous microorganisms by the scientific community working in the field of bio-lipids.
    Mots-clés : ATR FT-IR, attenuated total reflection Fourier transform, Biofuel, gas chromatography–mass spectrometry, GC-MS, infrared spectroscopy, lipid quantification, MESMIC, MICROBIO, Streptomyces, triacylglycerol.


  • L. Yan, Q. Zhang, M. - J. Virolle, et D. Xu, « In conditions of over-expression, WblI, a WhiB-like transcriptional regulator, has a positive impact on the weak antibiotic production of Streptomyces lividans TK24 », PLOS ONE, vol. 12, nᵒ 3, p. e0174781, mars 2017.

2016


  • I. Boukhris, T. Dulermo, H. Chouayekh, et M. - J. Virolle, « Evidence for the negative regulation of phytase gene expression in Streptomyces lividans and Streptomyces coelicolor », Journal of Basic Microbiology, vol. 56, nᵒ 1, p. 59-66, janv. 2016.
    Résumé : Sco7697, a gene encoding a phytase, enzyme able to degrade phytate (myo-inositol 1,2,3,4,5,6-hexakis phosphate), the most abundant phosphorus storing compound in plants is present in the genome of S. coelicolor, a soil born bacteria with a saprophytic lifestyle. The expression of this gene was previously shown to be induced in conditions of Pi limitation by the response regulator PhoP binding to an operator sequence, the PHO box, located upstream of the -35 promoter sequence. A close examination of the promoter region of sco7697 revealed the presence of another putative operator site, a Direct Repeat (DR), located downstream of the -10 promoter sequence. In order to determine whether this DR played a role in regulation of sco7697 expression, different variants of the phytase gene promoter region were transcriptionally fused to the ß-glucuronidase reporter gene (GUS). As expected, deletion of the PHO box led to abolition of sco7697 induction in conditions of Pi limitation. Interestingly, alteration of the DR correlated with a dramatic increase of GUS expression but only when PhoP was present. These results demonstrated that this DR is the site of strong negative regulation by an unknown repressor. The latter would impede the necessary activation of phytase expression by PhoP.
    Mots-clés : 6-Phytase, Bacterial Proteins, Base Sequence, Beta-glucuronidase reporter gene, Gene Expression Regulation, Bacterial, Glucuronidase, MESMIC, MICROBIO, Negative, Operon, PHO box, PhoP, PhoR, Phosphate limitation, Phytic Acid, Positive regulation, Promoter Regions, Genetic, Protein Binding, Repetitive Sequences, Nucleic Acid, Sequence Deletion, Soil Microbiology, Streptomyces coelicolor, Streptomyces lividans, Streptomyces phytase.

  • I. Boukhris, A. Farhat-Khemakhem, K. Bouchaala, M. - J. Virolle, et H. Chouayekh, « Cloning and characterization of the first actinomycete β-propeller phytase from Streptomyces sp. US42 », Journal of Basic Microbiology, vol. 56, nᵒ 10, p. 1080-1089, oct. 2016.
    Résumé : A gene encoding an extracellular phytase was cloned for the first time from an Actinomycete, Streptomyces sp. US42 and sequenced. The sequence of this gene revealed an encoded polypeptide (PHY US42) exhibiting one and six residues difference with the putative phytases of Streptomyces lividans TK24 and Streptomyces coelicolor A3(2), respectively. The molecular modeling of PHY US42 indicated that this phytase belongs to the group of β-propeller phytases that are usually calcium-dependent. PHY US42 was purified and characterized. Its activity was calcium-dependent and maximal at pH 7 and 65 °C. The enzyme was perfectly stable at pH ranging from 5 to 10 and its thermostability was greatly enhanced in the presence of calcium. Indeed, PHY US42 maintained 80% of activity after 10 min of incubation at 75 °C in the presence of 5 mM CaCl2 . PHY US42 was also found to exhibit high stability after incubation at 37 °C for 1 h in the presence of bovine bile and digestive proteases like of pepsin, trypsin, and chymotrypsin. Considering its biochemical properties, PHY US42 could be used as feed additive in combination with an acid phytase for monogastric animals.
    Mots-clés : 6-Phytase, Amino Acid Sequence, Base Sequence, Calcium, Calcium-dependent, Cloning, Molecular, DNA, Fungal, Enzyme Stability, Feed additive, MESMIC, MICROBIO, Sequence Analysis, DNA, Streptomyces, Substrate Specificity, β-propeller phytase.


  • T. Dulermo, F. Coze, M. - J. Virolle, V. Méchin, S. Baumberger, et M. Froissard, « Bioconversion of agricultural lignocellulosic residues into branched-chain fatty acids using <i>Streptomyces lividans</i> », OCL, vol. 23, nᵒ 2, p. A202, 2016.


  • M. - J. Virolle, A. Millan-Oropeza, C. Esnault, A. Smirnov, T. Dulermo, et A. Askora, « In Streptomyces, the switch between primary and secondary metabolism is underpinned by a transition from glycolytic to oxidative metabolism », New Biotechnology, vol. 33, p. S59-S60, 2016.


  • P. Vitry, R. Rebois, E. Bourillot, A. Deniset-Besseau, M. - J. Virolle, E. Lesniewska, et A. Dazzi, « Combining infrared and mode synthesizing atomic force microscopy: Application to the study of lipid vesicles inside Streptomyces bacteria », Nano Research, vol. 9, nᵒ 6, p. 1674-1681, 2016.

2015

2014

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