The Epipeptide Biosynthesis Locus epeXEPAB Is Widely Distributed in Firmicutes and Triggers Intrinsic Cell Envelope Stress

The epeXEPAB (formerly yydFGHIJ) locus of Bacillus subtilis encodes a minimalistic biosynthetic pathway for a linear antimicrobial epipeptide, EpeX, which is ribosomally produced and post-translationally processed by the action of the radical-SAM epimerase, EpeE, and a membrane-anchored signal 2 peptide peptidase, EpeP. The ABC transporter EpeAB provides intrinsic immunity against self-produced EpeX, without conferring resistance against extrinsically added EpeX. EpeX specifically targets, and severely perturbs the integrity of the cytoplasmic membrane, which leads to the induction of the Lia-dependent envelope stress response. Here, we provide new insights into the distribution, expression, and regulation of the minimalistic epeXEPAB locus of B. subtilis, as well as the biosynthesis and biological efficiency of the produced epipeptide EpeX*. A comprehensive comparative genomics study demonstrates that the epe-locus is restricted to but widely distributed within the phylum Firmicutes. The gene products of epeXEP are necessary and sufficient for the production of the mature antimicrobial peptide EpeX*. In B. subtilis, the epeXEPAB locus is transcribed from three different promoters, one upstream of epeX (PepeX) and two within epeP (PepeA1 and PepeA2). While the latter two are mostly constitutive, PepeX shows a growth phase-dependent induction at the onset of stationary phase. We demonstrate that this regulation is the result of the antagonistic action of two global regulators: The transition state regulator AbrB keeps the epe locus shut off during exponential growth by direct binding. This tight repression is relieved by the master regulator of sporulation, Spo0A, which counteracts the AbrB-dependent repression of epeXEPAB expression during the transition to stationary phase. The net result of these three ­promoters is an expression pattern that ensures EpeAB-dependent autoimmunity prior to EpeX* production. In the absence of EpeAB, the general envelope stress response proteins LiaIH can compensate for the loss of specific autoimmunity by providing sufficient protection against the membrane-perturbating action of EpeX*. Hence, the transcriptional regulation of epe expression and the resulting intrinsic induction of the two corresponding resistance functions, encoded by epeAB and liaIH, are well balanced to provide a need-based immunity against mature EpeX*.Peer Reviewe

Direct repair of a synthetic 5S-configured spore photoproduct by a spore photoproduct lyase.

International audienceThe spore photoproduct lyase is a Fe-S/AdoMet DNA repair enzyme, which directly repairs spore lesions, induced by UV irradiation of spores, using an unknown radical mechanism. The air sensitive radical SAM enzyme was for the first time challenged with synthetically pure substrates. It was found that the enzyme recognizes a synthetic 5S-configured spore lesion without the central phosphodiester bond. The 5R-configured lesion is in contrast to current belief not a substrate

Biosynthesis of the sactipeptide Ruminococcin C by the human microbiome: Mechanistic insights into thioether bond formation by radical SAM enzymes

Despite its major importance in human health, the metabolic potential of the human gut microbiota is still poorly understood. We have recently shown that biosynthesis of Ruminococcin C (RumC), a novel ribosomally synthesized and posttranslationally modified peptide (RiPP) produced by the commensal bacterium Ruminococcus gnavus, requires two radical SAM enzymes (RumMC1 and RumMC2) catalyzing the formation of four C-alpha-thioether bridges. These bridges, which are essential for RumC's antibiotic properties against human pathogens such as Clostridium perfringens, define two hairpin domains giving this sactipeptide (sulfur-to-alpha-carbon thioether-containing peptide) an unusual architecture among natural products. We report here the biochemical and spectroscopic characterizations of RumMC2. EPR spectroscopy and mutagenesis data support that RumMC2 is a member of the large family of SPASM domain radical SAM enzymes characterized by the presence of three [4Fe-4S] clusters. We also demonstrate that this enzyme initiates its reaction by C-alpha H-atom abstraction and is able to catalyze the formation of nonnatural thioether bonds in engineered peptide substrates. Unexpectedly, our data support the formation of a ketoimine rather than an alpha,beta-dehydro-amino acid intermediate during C-alpha-thioether bridge LC-MS/MS fragmentation. Finally, we explored the roles of the leader peptide and of the RiPP precursor peptide recognition element, present in myriad RiPP-modifying enzymes. Collectively, our data support a more complex role for the peptide recognition element and the core peptide for the installation of posttranslational modifications in RiPPs than previously anticipated and suggest a possible reaction intermediate for thioether bond formation

Extracorporeal Membrane Oxygenation for Severe Acute Respiratory Distress Syndrome associated with COVID-19: An Emulated Target Trial Analysis.

RATIONALE: Whether COVID patients may benefit from extracorporeal membrane oxygenation (ECMO) compared with conventional invasive mechanical ventilation (IMV) remains unknown. OBJECTIVES: To estimate the effect of ECMO on 90-Day mortality vs IMV only Methods: Among 4,244 critically ill adult patients with COVID-19 included in a multicenter cohort study, we emulated a target trial comparing the treatment strategies of initiating ECMO vs. no ECMO within 7 days of IMV in patients with severe acute respiratory distress syndrome (PaO2/FiO2 <80 or PaCO2 ≥60 mmHg). We controlled for confounding using a multivariable Cox model based on predefined variables. MAIN RESULTS: 1,235 patients met the full eligibility criteria for the emulated trial, among whom 164 patients initiated ECMO. The ECMO strategy had a higher survival probability at Day-7 from the onset of eligibility criteria (87% vs 83%, risk difference: 4%, 95% CI 0;9%) which decreased during follow-up (survival at Day-90: 63% vs 65%, risk difference: -2%, 95% CI -10;5%). However, ECMO was associated with higher survival when performed in high-volume ECMO centers or in regions where a specific ECMO network organization was set up to handle high demand, and when initiated within the first 4 days of MV and in profoundly hypoxemic patients. CONCLUSIONS: In an emulated trial based on a nationwide COVID-19 cohort, we found differential survival over time of an ECMO compared with a no-ECMO strategy. However, ECMO was consistently associated with better outcomes when performed in high-volume centers and in regions with ECMO capacities specifically organized to handle high demand. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/)

A missed Fe-S cluster handoff causes a metabolic shakeup

The general framework of pathways by which iron-sulfur (Fe-S) clusters are assembled in cells is well-known, but the cellular consequences of disruptions to that framework are not fully understood. Crooks et al. report a novel cellular system that creates an acute Fe-S cluster deficiency, using mutants of ISCU, the main scaffold protein for Fe-S cluster assembly. Surprisingly, the resultant metabolic reprogramming leads to the accumulation of lipid droplets, a situation encountered in many poorly understood pathological conditions, highlighting unanticipated links between Fe-S assembly machinery and human disease

Intérêt du cardiofréquencemètre chez le coureur de fond

DIJON-BU Médecine Pharmacie (212312103) / SudocSudocFranceF

Développement d'outils enzymatiques pour la caractérisation du fucoïdane extrait de l'algue Ascophyllum nodosum (purification et propriétés de nouvelles hydrolases isolées du mollusque marin Pecten maximus)

PARIS13-BU Sciences (930792102) / SudocSudocFranceF

DNA repair by the radical SAM enzyme spore Photoproduct Lyase: from biochemistry to structural investigations

Radical S-adenosyl-L-methionine (SAM) enzymes have emerged as one of the last superfamilies of enzymes discovered to date. Arguably, it is the most versatile group of enzymes involved in at least 85 biochemical transformations. One of the founding members of this enzyme superfamily is the spore photoproduct (SP) lyase, a DNA repair enzyme catalyzing the direct reversal repair of a unique DNA lesion, the so-called spore photoproduct, back into two thymidine residues. Discovered more than 20 years ago in the bacterium Bacillus subtilis, SP lyase has been shown to be widespread in the endospore-forming Firmicutes from the Bacilli and Clostridia classes and to use radical-based chemistry to perform C-C bond breakage, a chemically challenging reaction. This review describes how the work on SP lyase has illuminated a unique strategy for DNA repair and provided major advances in our understanding of the emerging radical SAM superfamily of enzymes, from a biochemical and structural perspective

Radical SAM Enzymes in the Biosynthesis of Ribosomally Synthesized and Post-translationally Modified Peptides (RiPPs)

Ribosomally-synthesized and post-translationally modified peptides (RiPPs) are a large and diverse family of natural products. They possess interesting biological properties such as antibiotic or anticancer activities, making them attractive for therapeutic applications. In contrast to polyketides and non-ribosomal peptides, RiPPs derive from ribosomal peptides and are post-translationally modified by diverse enzyme families. Among them, the emerging superfamily of radical SAM enzymes has been shown to play a major role. These enzymes catalyze the formation of a wide range of post-translational modifications some of them having no counterparts in living systems or synthetic chemistry. The investigation of radical SAM enzymes has not only illuminated unprecedented strategies used by living systems to tailor peptides into complex natural products but has also allowed to uncover novel RiPP families. In this review, we summarize the current knowledge on radical SAM enzymes catalyzing RiPP post-translational modifications and discuss their mechanisms and growing importance notably in the context of the human microbiota