Escherichia coli RuvBL268S: A mutant RuvB protein that exhibits wild-type activities in vitro but confers a UV-sensitive ruv phenotype in vivo

The RuvABC proteins of Escherichia coli process recombination intermediates during genetic recombination and DNA repair. RuvA and RuvB promote branch migration of Holliday junctions, a process that extends heteroduplex DNA. Together with RuvC, they form a RuvABC complex capable of Holliday junction resolution. Branch migration by RuvAB is mediated by RuvB, a hexameric ring protein that acts as an ATP-driven molecular pump. To gain insight into the mechanism of branch migration, random mutations were introduced into the ruvB gene by PCR and a collection of mutant alleles were obtained. Mutation of leucine 268 to serine resulted in a severe UV-sensitive phenotype, characteristic of a ruv defect. Here, we report a biochemical analysis of the mutant protein RuvBL268S. Unexpectedly, the purified protein is fully active in vitro with regard to its ATPase, DNA binding and DNA unwinding activities. It also promotes efficient branch migration in combination with RuvA, and forms functional RuvABC-Holliday junction resolvase complexes. These results indicate that RuvB may perform some additional, and as yet undefined, function that is necessary for cell survival after UV-irradiatio

The Mycobacterium Tuberculosis FAS-II Dehydratases and Methyltransferases Define the Specificity of the Mycolic Acid Elongation Complexes

BACKGROUND: The human pathogen Mycobacterium tuberculosis (Mtb) has the originality of possessing a multifunctional mega-enzyme FAS-I (Fatty Acid Synthase-I), together with a multi-protein FAS-II system, to carry out the biosynthesis of common and of specific long chain fatty acids: the mycolic acids (MA). MA are the main constituents of the external mycomembrane that represents a tight permeability barrier involved in the pathogenicity of Mtb. The MA biosynthesis pathway is essential and contains targets for efficient antibiotics. We have demonstrated previously that proteins of FAS-II interact specifically to form specialized and interconnected complexes. This finding suggested that the organization of FAS-II resemble to the architecture of multifunctional mega-enzyme like the mammalian mFAS-I, which is devoted to the fatty acid biosynthesis. PRINCIPAL FINDINGS: Based on conventional and reliable studies using yeast-two hybrid, yeast-three-hybrid and in vitro Co-immunoprecipitation, we completed here the analysis of the composition and architecture of the interactome between the known components of the Mtb FAS-II complexes. We showed that the recently identified dehydratases HadAB and HadBC are part of the FAS-II elongation complexes and may represent a specific link between the core of FAS-II and the condensing enzymes of the system. By testing four additional methyltransferases involved in the biosynthesis of mycolic acids, we demonstrated that they display specific interactions with each type of complexes suggesting their coordinated action during MA elongation. SIGNIFICANCE: These results provide a global update of the architecture and organization of a FAS-II system. The FAS-II system of Mtb is organized in specialized interconnected complexes and the specificity of each elongation complex is given by preferential interactions between condensing enzymes and dehydratase heterodimers. This study will probably allow defining essential and specific interactions that correspond to promising targets for Mtb FAS-II inhibitors

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research

Bacterial Cell Envelopes from Monoderms to Diderms: Composition, Architecture, and Origin

International audienceThe bacterial envelope is a complex, multi-layered structure, whose primary function is to protect the cell from environmental threats. For over a century, the Gram-staining method has been a foundational technique for classifying bacteria, distinguishing between Gram-positive bacteria, which retain the stain, and Gram-negative bacteria, which do not. Historically, prokaryotes have been classified in 41 accepted phyla of Bacteria and 16 phyla of Archaea, a system that overlooks the nuances revealed by Gram-staining properties. Advances in genomic sequencing have significantly reshaped our understanding of bacterial phylogeny. Additionally, insights into the genetics of cell envelope biogenesis have provided a new perspective on the bacterial Tree of Life. Bacterial envelopes are now categorized based on the number of membranes they possess, classifying cells as either diderms or monoderms. The presence or absence of lipopolysaccharides (LPS) in diderm envelopes is a critical distinguishing factor. This chapter provides a concise overview of the current knowledge regarding the composition and architecture of bacterial envelopes. It highlights how diverse structural adaptations serve a common purpose: ensuring the survival and protection of bacteria in various environments

Expression genique de la sequence d'insertion IS1 : mise en evidence et caracterisation d'une proteine indispensable a la transposition

SIGLECNRS T Bordereau / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Recherche et étude des gènes de glycosyl-transférases impliqués dans la biosynthèse de constituants de l'enveloppe mycobactérienne

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Architecture et essentialité des complexes de biosynthèse des acides mycoliques de la bactérie pathogène Mycobacterium tuberculosis

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Cell Membrane Electropermeabilization

The behavior of cell membranes can be strongly affected when pulsed electric fields are delivered on a cell culture. Fields can be applied either continuously or during a short duration (electric pulse). Electropulsation results in the cell membrane permeabilization. Electropermeabilization is one of the most successful methods to introduce foreign molecules in living cells in vitro or to assist the extraction of high value compounds from microorganisms and from plant cells. The treatment is observed to be lethal. Death of the organisms is not due to the products of electrolysis; the temperature rise of the suspension is too small and short-lived to cause lethality. Many physical effects of the field pulse can be observed in the cell. From the dielectric properties of the cell membrane, it is proposed that the field modulates the transmembrane potential of cells. The induced potential triggers conformational changes in the membrane structure when it is larger than a critical value. These structural changes result in the observed loss of the cell's permeability barrier properties. The level of membrane alteration is observed to increase with the pulse length and number of pulses, and by the field strength in the suspension. The molecular processes are complicated and remain poorly characterized. Membrane damage is demonstrated by the lysis of protoplasts, the leakage of intracellular contents, and the loss of the ability of Escherichia coli to plasmolyze in a hypertonic medium

Etude des interactions protéine-protéine au sein d'un processus vital (la biosynthèse des acides mycoliques chez Mycobacterium tuberculosis)

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF