DOCSLIB.ORG

The Identification of Genes and Enzymes in the Aldoxime-Nitrile Metabolising Pathway

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
The Identification of Genes and Enzymes in the Aldoxime-Nitrile Metabolising Pathway THE IDENTIFICATION OF GENES AND ENZYMES IN THE ALDOXIME-NITRILE METABOLISING PATHWAY Submitted to Waterford Institute of Technology for the Degree of Doctor of Philosophy By Tríona-Marie Dooley-Cullinane Pharmaceutical and Molecular Biotechnology Research Center (PMBRC) Waterford Institute of Technology, Waterford, Ireland Under the Supervision of Dr. Lee Coffey and Dr. Catherine O’Reilly 2019 DECLARATION The work described in this thesis isoriginal and was solely carried out by the author, and the work of others has been duly referenced in the text. No part of this thesis has been previously submitted for a degree at this or any other institute. Signed:_____________________ Date:_______________________ i ACKNOWLEDGEMENTS I owe a special thanks to my supervisors Dr. Lee Coffey and Dr. Catherine O’Reilly. I was fortunate to have been taught by both Lee and Catherine as an undergraduate student and their love of science and research was nothing short of inspiring. Thanks to both Catherine and Lee for your advice and support while I constructed this thesis, and for helping me find my way through the difficult days and months of writing. I would like to thank the technical staff of WIT, in particular Mari, Walter, Aidan, Pat and Stephen. In times of need, you were always on hand and I will forever be grateful for the guidance and advice. I am also indebted to my friends and fellow postgraduate students at WIT. Hazal thanks for always being a source of laughter and to Caio, your company in B19 made the long hours in the lab full of fun. I owe a special thanks to both Andrew and Caroline for the days of unwavering support, laughter and copious amounts of caffeine. They do say laughter is the best form of medicine. As the saying goes, your friends are the family you choose. If these past years have taught me anything, they have further instilled in me that I have chosen wisely. To both Sarah C and Sarah B, the endless days and nights of fun, dance parties and most importantly tea parties have gotten me through some of the hardest times. I will forever be grateful to you both. To my family, I cannot thank you enough for your patience, support and most importantly love. To my parents, you have both been amazing role models, showing that good work ethic and perseverance are the key to achieving and succeeding in life. Dad, I owe you a special thanks for showing me the importance of never giving up and for always giving advice and support. Mam, you have always believed in me and always been a source of encouragement and support, more than you realise. All I can hope is that one day, I become as well rounded and successful as you both, in all aspects of my life. Aoife, you helped to make the hardest days easier, I will be forever grateful for your company and sense of humor, you always knew when I needed it most. To my second family, the ii Gannon-Stephenson’s, you have made me feel like part of the furniture over the last nine years and have provided more support that you will ever know, I will be forever thankful. To Marcus, I don’t know where to begin. You have been on this journey with me since my undergraduate studies. You have witnessed all the ups and downs and your support and love has never waivered throughout. You have been there every day and night through it all and knew exactly what I needed, even when I didn’t. Your sense of humor and wit have made the longest days more bearable. I will forever be indebted to you for your years of guidance, support, patience and love. Finally, to Alice, this dissertation is dedicated to your memory. I hope that one day I can become half the woman you were, and I thank you for your encouragement and support during my younger years. iii ABSTRACT Biocatalysis is gaining much attention for the production of pharmaceutical intermediates, drug compounds and, fine chemicals. Biocatalysis, as a route of synthesis, boasts many benefits over traditional chemical synthesis. It can account for enantiopure compounds, lower waste production, decreases the need for potential purification and down-stream processing while also allowing the reactions to be carried out at milder conditions, such as lower temperatures and a relatively neutral pH (Gong et al. 2012, Singh et al. 2006). Due to the often low efficacy of racemic drugs or in fact the side effects caused by one of the enantiomers, enantiopure products are highly sought after (Albarrán-Velo et al. 2018). Thalidomide, a drug that was previously prescribed to treat morning sickness during pregnancy was sold as a safe and effective anti-emetic, in its racemic form. Unfortunately, years later the level of incidents of miscarriage and foetal birth defects observed amongst the women who were prescribed the drug led to the discovery that the S-enantiomer of the drug was teratogenic. Enzymes comprising the Aldoxime-Nitrile pathway are the focus of the body of work presented in this thesis. In 2010, it was reported that there were over 30 nitrile-containing pharmaceuticals on the market with another 20 at clinical trial stage (Fleming et al. 2010). Many nitrile containing drugs or intermediates may have been produced via chemical synthesis coupled with biocatalysis. Focusing on the identification of aldoxime dehydratase enzymes can offer a new, improved route to nitrile synthesis. The research presented in chapter two and published by (Dooley-Cullinane et al. 2016) presents a new high-throughput real-time (qPCR) for the detection of novel aldoxime dehydratase genes. The potential application of these genes/enzymes was reinforced by the Food and Drug Administration (FDA) approval of 11 nitrile containing pharmaceuticals since 2010. This is an indication as to what is to come with regard to an increase in market size for bio- pharmaceuticals produced by biocatalysis, in particular nitrile containing pharmaceuticals. The remaining enzymes which comprise the aldoxime-nitrile pathway, the nitrile- degrading enzymes such as nitrilases and nitrile hydratases (NHases), have been shown iv to display a broad substrate scope and complex nitrile substrates can be converted into their corresponding amides with the final enzyme, and amidase converting the amide to the corresponding carboxylic acids. The area of nitrile metabolism has been of great interest and will be into the foreseeable future (Huisman and Collier 2013, Gong et al. 2012). The body of work presented, details outputs and discoveries which form a part of the larger web of research undertaken by the molecular biocatalysis research group. The overall aim of research carried out by the group is the identification and discovery of novel genes and enzymes towards the production of industrially or biopharmaceutical relevant products. This thesis presents two novel molecular methods for the detection of genes within the aldoxime-nitrile pathway. A novel qPCR method was designed for the detection of aldoxime dehydratase genes. The second molecular method presented is a novel clade-specific touchdown conventional PCR which allows for the clade specific detection of novel nitrilase genes. Additionally, this protocol allows for the detection of nitrilase genes with predicted enzymatic activity in model substrates based on their clade classification. A novel nitrilase previously identified from Burkholderia sp. LC8 was cloned, expressed and characterised with results displaying a broad substrate range and excellent enantioselectivity towards mandelonitrile. The research also documents the discovery of a novel nitrilase partial sequence through the construction of a genomic fosmid library from genomic DNA of Rhodococcus erythropolis SET-1. This body of work involved the screening of fosmid clones for functional activity on 3-HGN. Further work presented a discussion on the attempts to elucidate the full nitrilase gene sequence via fosmid insert sequencing and also whole genome sequencing which led to the identification of a nitrile hydratase/amidase genomic cluster in R. erythropolis SET-1. v TABLE OF CONTENTS Acknowledgements ....................................................................................................... ii Abstract ........................................................................................................................ iv Table of Contents ......................................................................................................... vi Proposed Research…………………………………………………………………….1 1 Introduction ............................................................................................................... 5 1.1 Nitriles and their occurrence .............................................................................. 5 1.2 Toxicity of nitriles .............................................................................................. 5 1.3 Nitrile degrading pathways ................................................................................ 6 1.4 Nitrile hydrolysis ................................................................................................ 6 1.5 Nitrile metabolising enzymes ............................................................................. 7 1.5.1 Nitrilases (EC 3.5.5.1) ................................................................................. 7 1.5.2 Nitrilase classification and structure ........................................................... 9 1.5.3 Nitrilase activity ........................................................................................ 13 1.5.4 Nitrilase gene structure ............................................................................
Load more

Recommended publications
  • The Cyanide Hydratase from Neurospora Crassa Forms a Helix Which Has a Dimeric Repeat
    Appl Microbiol Biotechnol (2009) 82:271–278 DOI 10.1007/s00253-008-1735-4 BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS The cyanide hydratase from Neurospora crassa forms a helix which has a dimeric repeat Kyle C. Dent & Brandon W. Weber & Michael J. Benedik & B. Trevor Sewell Received: 2 July 2008 /Revised: 24 September 2008 /Accepted: 25 September 2008 / Published online: 23 October 2008 # Springer-Verlag 2008 Abstract The fungal cyanide hydratases form a functionally Introduction specialized subset of the nitrilases which catalyze the hydrolysis of cyanide to formamide with high specificity. The generation of cyanide containing waste results from These hold great promise for the bioremediation of cyanide cyanide utilization in a variety of applications, spanning wastes. The low resolution (3.0 nm) three-dimensional such diverse industries as gold and silver mining, metal reconstruction of negatively stained recombinant cyanide electroplating, polymer synthesis, and the production of hydratase fibers from the saprophytic fungus Neurospora fine chemicals, pharmaceuticals, dyes, and agricultural crassa by iterative helical real space reconstruction reveals products (Baxter and Cummings 2006). Traditionally, that enzyme fibers display left-handed D1 S5.4 symmetry chemical oxidation or stabilizations are used to treat these with a helical rise of 1.36 nm. This arrangement differs from wastes; however, many groups have been studying the previously characterized microbial nitrilases which demon- potential for bioremediation of such cyanide waste strate a structure built along similar principles but with a (O’Reilly and Turner 2003; Jandhyala et al. 2005). reduced helical twist. The cyanide hydratase assembly is The most promising approach utilizes cyanide degrada- stabilized by two dyadic interactions between dimers across tion by enzymes belonging to the nitrilase family (Pace and the one-start helical groove.
    [Show full text]
  • Cyanide-Degrading Enzymes for Bioremediation A
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Texas A&M University CYANIDE-DEGRADING ENZYMES FOR BIOREMEDIATION A Thesis by LACY JAMEL BASILE Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE August 2008 Major Subject: Microbiology CYANIDE-DEGRADING ENZYMES FOR BIOREMEDIATION A Thesis by LACY JAMEL BASILE Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Approved by: Chair of Committee, Michael Benedik Committee Members, Susan Golden James Hu Wayne Versaw Head of Department, Vincent Cassone August 2008 Major Subject: Microbiology iii ABSTRACT Cyanide-Degrading Enzymes for Bioremediation. (August 2008) Lacy Jamel Basile, B.S., Texas A&M University Chair of Advisory Committee: Dr. Michael Benedik Cyanide-containing waste is an increasingly prevalent problem in today’s society. There are many applications that utilize cyanide, such as gold mining and electroplating, and these processes produce cyanide waste with varying conditions. Remediation of this waste is necessary to prevent contamination of soils and water. While there are a variety of processes being used, bioremediation is potentially a more cost effective alternative. A variety of fungal species are known to degrade cyanide through the action of cyanide hydratases, a specialized subset of nitrilases which hydrolyze cyanide to formamide. Here I report on previously unknown and uncharacterized nitrilases from Neurospora crassa, Gibberella zeae, and Aspergillus nidulans. Recombinant forms of four cyanide hydratases from N.
    [Show full text]
  • Purification and Characterization of a Novel Nitrilase of Rhodococcus
    JOURNAL OF BACTERIOLOGY, Sept. 1990, p. 4807-4815 Vol. 172, No. 9 0021-9193/90/094807-09$02.00/0 Copyright X3 1990, American Society for Microbiology Purification and Characterization of a Novel Nitrilase of Rhodococcus rhodochrous K22 That Acts on Aliphatic Nitriles MICHIHIKO KOBAYASHI,* NORIYUKI YANAKA, TORU NAGASAWA, AND HIDEAKI YAMADA Department ofAgricultural Chemistry, Faculty ofAgriculture, Kyoto University, Sakyo-ku, Kyoto 606, Japan Received 18 April 1990/Accepted 8 June 1990 A novel nitrilase that preferentially catalyzes the hydrolysis of aliphatic nitriles to the corresponding carboxylic acids and ammonia was found in the cells of a facultative crotononitrile-utilizing actinomycete isolated from soil. The strain was taxonomically studied and identified as Rhodococcus rhodochrous. The nitrilase was purified, with 9.08% overall recovery, through five steps from a cell extract of the stain. After the last step, the purified enzyme appeared to be homogeneous, as judged by polyacrylamide gel electrophoresis, analytical centrifugation, and double immunodiffusion in agarose. The relative molecular weight values for the native enzyme, estimated from the ultracentrifugal equilibrium and by high-performance liquid chromatog- raphy, were approximately 604,000 + 30,000 and 650,000, respectively, and the enzyme consisted of 15 to 16 subunits identical in molecular weight (41,000). The enzyme acted on aliphatic olefinic nitriles such as crotononitrile and acrylonitrile as the most suitable substrates. The apparent Km values for crotononitrile and acrylonitrile were 18.9 and 1.14 mM, respectively. The nitrilase also catalyzed the direct hydrolysis of saturated aliphatic nitriles, such as valeronitrile, 4-chlorobutyronitrile, and glutaronitrile, to the correspond- ing acids without the formation of amide intermediates.
    [Show full text]
  • Purification and Characterization of Nitrilase from Fusarium Solani
    Process Biochemistry 45 (2010) 1115–1120 Contents lists available at ScienceDirect Process Biochemistry journal homepage: www.elsevier.com/locate/procbio Purification and characterization of nitrilase from Fusarium solani IMI196840 Vojtechˇ Vejvoda a, David Kubácˇ a,Alzbˇ etaˇ Davidová a, Ondrejˇ Kaplan a, Miroslav Sulcˇ a,b, Ondrejˇ Svedaˇ a, Radka Chaloupková c, Ludmila Martínková a,∗ a Institute of Microbiology, Centre of Biocatalysis and Biotransformation, Academy of Sciences of the Czech Republic, Vídenskᡠ1083, CZ-142 20 Prague, Czech Republic b Department of Biochemistry, Faculty of Science, Charles University Prague, Hlavova 8, CZ-128 40 Prague, Czech Republic c Loschmidt Laboratories, Department of Experimental Biology and Centre of Biocatalysis and Biotransformation, Faculty of Science, Masaryk University, Kamenice 5/A4, CZ-625 00 Brno, Czech Republic article info abstract Article history: Nitrilase activity in Fusarium solani IMI196840 (approx. 1500 U l−1 of culture broth) was induced by 2- Received 11 November 2009 cyanopyridine. The enzyme was purified by a factor of 20.3 at a yield of 26.9%. According to gel filtration, Received in revised form 26 March 2010 the holoenzyme was an approx. 550-kDa homooligomer consisting of subunits with a molecular weight Accepted 30 March 2010 of approximately 40 kDa, as determined by SDS-PAGE. Mass spectrometry analysis of the tryptic frag- ments suggested a high similarity of this enzyme to the hypothetical CN hydrolases from Aspergillus Keywords: oryzae, Gibberella zeae, Gibberella moniliformis and Nectria haematococca. Circular dichroism and fluo- Fusarium solani rescence spectra indicated that secondary structure content and overall tertiary structure, respectively, Nitrilase were almost identical in nitrilases from F.
    [Show full text]
  • Structures, Functions, and Mechanisms of Filament Forming Enzymes: a Renaissance of Enzyme Filamentation
    Structures, Functions, and Mechanisms of Filament Forming Enzymes: A Renaissance of Enzyme Filamentation A Review By Chad K. Park & Nancy C. Horton Department of Molecular and Cellular Biology University of Arizona Tucson, AZ 85721 N. C. Horton ([email protected], ORCID: 0000-0003-2710-8284) C. K. Park ([email protected], ORCID: 0000-0003-1089-9091) Keywords: Enzyme, Regulation, DNA binding, Nuclease, Run-On Oligomerization, self-association 1 Abstract Filament formation by non-cytoskeletal enzymes has been known for decades, yet only relatively recently has its wide-spread role in enzyme regulation and biology come to be appreciated. This comprehensive review summarizes what is known for each enzyme confirmed to form filamentous structures in vitro, and for the many that are known only to form large self-assemblies within cells. For some enzymes, studies describing both the in vitro filamentous structures and cellular self-assembly formation are also known and described. Special attention is paid to the detailed structures of each type of enzyme filament, as well as the roles the structures play in enzyme regulation and in biology. Where it is known or hypothesized, the advantages conferred by enzyme filamentation are reviewed. Finally, the similarities, differences, and comparison to the SgrAI system are also highlighted. 2 Contents INTRODUCTION…………………………………………………………..4 STRUCTURALLY CHARACTERIZED ENZYME FILAMENTS…….5 Acetyl CoA Carboxylase (ACC)……………………………………………………………………5 Phosphofructokinase (PFK)……………………………………………………………………….6
    [Show full text]
  • Changes in the Sclerotinia Sclerotiorum Transcriptome During Infection of Brassica Napus
    Seifbarghi et al. BMC Genomics (2017) 18:266 DOI 10.1186/s12864-017-3642-5 RESEARCHARTICLE Open Access Changes in the Sclerotinia sclerotiorum transcriptome during infection of Brassica napus Shirin Seifbarghi1,2, M. Hossein Borhan1, Yangdou Wei2, Cathy Coutu1, Stephen J. Robinson1 and Dwayne D. Hegedus1,3* Abstract Background: Sclerotinia sclerotiorum causes stem rot in Brassica napus, which leads to lodging and severe yield losses. Although recent studies have explored significant progress in the characterization of individual S. sclerotiorum pathogenicity factors, a gap exists in profiling gene expression throughout the course of S. sclerotiorum infection on a host plant. In this study, RNA-Seq analysis was performed with focus on the events occurring through the early (1 h) to the middle (48 h) stages of infection. Results: Transcript analysis revealed the temporal pattern and amplitude of the deployment of genes associated with aspects of pathogenicity or virulence during the course of S. sclerotiorum infection on Brassica napus. These genes were categorized into eight functional groups: hydrolytic enzymes, secondary metabolites, detoxification, signaling, development, secreted effectors, oxalic acid and reactive oxygen species production. The induction patterns of nearly all of these genes agreed with their predicted functions. Principal component analysis delineated gene expression patterns that signified transitions between pathogenic phases, namely host penetration, ramification and necrotic stages, and provided evidence for the occurrence of a brief biotrophic phase soon after host penetration. Conclusions: The current observations support the notion that S. sclerotiorum deploys an array of factors and complex strategies to facilitate host colonization and mitigate host defenses. This investigation provides a broad overview of the sequential expression of virulence/pathogenicity-associated genes during infection of B.
    [Show full text]
  • Comparison of Nitrile Hydratases in Rhodococcus Rhodochrous DAP 96253 and DAP 96622 Growing on Inducing and Non- Inducing Media
    Georgia State University ScholarWorks @ Georgia State University Biology Dissertations Department of Biology Spring 4-26-2013 Comparison of Nitrile Hydratases in Rhodococcus Rhodochrous DAP 96253 and DAP 96622 Growing on Inducing and Non- Inducing Media Fengkun Du Georgia State University Follow this and additional works at: https://scholarworks.gsu.edu/biology_diss Recommended Citation Du, Fengkun, "Comparison of Nitrile Hydratases in Rhodococcus Rhodochrous DAP 96253 and DAP 96622 Growing on Inducing and Non-Inducing Media." Dissertation, Georgia State University, 2013. https://scholarworks.gsu.edu/biology_diss/130 This Dissertation is brought to you for free and open access by the Department of Biology at ScholarWorks @ Georgia State University. It has been accepted for inclusion in Biology Dissertations by an authorized administrator of ScholarWorks @ Georgia State University. For more information, please contact [email protected]. COMPARISON OF NITRILE HYDRATASES IN RHODOCOCCUS RHODOCHROUS DAP 96253 AND DAP 96622 GROWING ON INDUCING AND NON INDUCING MEDIA by FENGKUN DU Under the Direction of George E. Pierce ABSTRACT Nitrile hydratase activity in Rhodococcus rhodochrous DAP 96253 can be induced with multiple inducers that include urea, cobalt (Co), iron (Fe) and nickel (Ni). When induced with Co/urea, cells of R. rhodochrous DAP 96253 expressed the highest level of nitrile hydratase activity (~200 units/min·mg-cdw) when compared with the other inducers tested. Cells induced with Co had the second highest nitrile hydratase activity (~7 units/min·mg-cdw), whereas in the uninduced cells, nitrile hydratase activity was lower than 1 unit/min·mg-cdw. Similarly in R. rhodochrous DAP 96622, when induced with Co/urea, the nitrile hydratase activity of R.
    [Show full text]
  • Investigation of Iron's Role in Alginate Production And
    INVESTIGATION OF IRON’S ROLE IN ALGINATE PRODUCTION AND MUCOIDY BY PSEUDOMONAS AERUGINOSA by JACINTA ROSE SCHRAUFNAGEL B.S., University of Colorado Denver, 2003 M.S., University of Colorado Denver, 2006 A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Doctor of Philosophy Molecular Biology Program 2013 This thesis for the Doctor of Philosophy degree by Jacinta Rose Schraufnagel has been approved for the Molecular Biology Program by David Barton, Chair Michael Vasil, Advisor Mair Churchill Jerry Schaack Michael Schurr Martin Voskuil Date 10/30/13 ii Schraufnagel, Jacinta R. (Ph.D., Molecular Biology) Investigation of Iron’s Role in Alginate Production and Mucoidy by Pseudomonas aeruginosa. Thesis directed by Professor Michael Vasil. ABSTRACT Iron, an essential nutrient, influences the production of major virulence factors and the development of structured non-alginate biofilms in Pseudomonas aeruginosa. Fur, pyoverdine and various concentrations of iron sources have all been shown to affect non-alginate biofilm formation. Remarkably however, the role of iron in alginate biofilm formation and mucoidy is unknown. Because of alginate’s association with biofilms in chronic cystic fibrosis (CF) lung infections, we examined the influence of iron on alginate production and mucoidy in P. aeruginosa. Herein, we identify and characterize a novel iron-regulated protein that affects the transcription of alginate biosynthesis genes. Further, contrary to what is known about non-alginate biofilms, we discovered that biologically relevant concentrations of iron (10 µM – 300 µM), in various forms (e.g. FeCl3, Ferric Citrate, Heme), led to the decreased expression of alginate biosynthesis genes and the dispersion of alginate biofilms in mucoid P.
    [Show full text]
  • Diversity of Antisense and Other Non-Coding Rnas in Archaea Revealed by Comparative Small RNA Sequencing in Four Pyrobaculum Species
    Lawrence Berkeley National Laboratory Recent Work Title Diversity of Antisense and Other Non-Coding RNAs in Archaea Revealed by Comparative Small RNA Sequencing in Four Pyrobaculum Species. Permalink https://escholarship.org/uc/item/94r6n6t1 Journal Frontiers in microbiology, 3(JUL) ISSN 1664-302X Authors Bernick, David L Dennis, Patrick P Lui, Lauren M et al. Publication Date 2012 DOI 10.3389/fmicb.2012.00231 Peer reviewed eScholarship.org Powered by the California Digital Library University of California ORIGINAL RESEARCH ARTICLE published: 02 July 2012 doi: 10.3389/fmicb.2012.00231 Diversity of antisense and other non-coding RNAs in archaea revealed by comparative small RNA sequencing in four Pyrobaculum species David L. Bernick 1, Patrick P.Dennis 2, Lauren M. Lui 1 andTodd M. Lowe 1* 1 Department of Biomolecular Engineering, University of California, Santa Cruz, CA, USA 2 Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA Edited by: A great diversity of small, non-coding RNA (ncRNA) molecules with roles in gene regula- Frank T. Robb, University of California, tion and RNA processing have been intensely studied in eukaryotic and bacterial model USA organisms, yet our knowledge of possible parallel roles for small RNAs (sRNA) in archaea Reviewed by: Mircea Podar, Oak Ridge National is limited. We employed RNA-seq to identify novel sRNA across multiple species of the Laboratory, USA hyperthermophilic genus Pyrobaculum, known for unusual RNA gene characteristics. By Imke Schroeder, University of comparing transcriptional data collected in parallel among four species, we were able to California Los Angeles, USA identify conserved RNA genes fitting into known and novel families.
    [Show full text]
  • Rnaseq Analysis of Aspergillus Fumigatus in Blood Reveals a Just Wait and See Resting Stage Behavior
    Irmer et al. BMC Genomics (2015) 16:640 DOI 10.1186/s12864-015-1853-1 RESEARCH ARTICLE Open Access RNAseq analysis of Aspergillus fumigatus in blood reveals a just wait and see resting stage behavior Henriette Irmer1, Sonia Tarazona2, Christoph Sasse1, Patrick Olbermann4, Jürgen Loeffler5, Sven Krappmann4,6, Ana Conesa2,3 and Gerhard H. Braus1* Abstract Background: Invasive aspergillosis is started after germination of Aspergillus fumigatus conidia that are inhaled by susceptible individuals. Fungal hyphae can grow in the lung through the epithelial tissue and disseminate hematogenously to invade into other organs. Low fungaemia indicates that fungal elements do not reside in the bloodstream for long. Results: We analyzed whether blood represents a hostile environment to which the physiology of A. fumigatus has to adapt. An in vitro model of A. fumigatus infection was established by incubating mycelium in blood. Our model allowed to discern the changes of the gene expression profile of A. fumigatus at various stages of the infection. The majority of described virulence factors that are connected to pulmonary infections appeared not to be activated during the blood phase. Three active processes were identified that presumably help the fungus to survive the blood environment in an advanced phase of the infection: iron homeostasis, secondary metabolism, and the formation of detoxifying enzymes. Conclusions: We propose that A. fumigatus is hardly able to propagate in blood. After an early stage of sensing the environment, virtually all uptake mechanisms and energy-consuming metabolic pathways are shut-down. The fungus appears to adapt by trans-differentiation into a resting mycelial stage.
    [Show full text]
  • Source: the Arabidopsis Information Resource (TAIR);
    Table S1 List of targeted loci and information about their function in Arabidopsis thaliana (source: The Arabidopsis Information Resource (TAIR); https://www.arabidopsis.org/tools/bulk/genes/index.jsp). Locus Gene Model Gene Model Description Gene Model Primary Gene Symbol All Gene Symbols Identifier Name Type AT1G78800 AT1G78800.1 UDP-Glycosyltransferase superfamily protein_coding protein;(source:Araport11) AT5G06830 AT5G06830.1 hypothetical protein;(source:Araport11) protein_coding AT2G31740 AT2G31740.1 S-adenosyl-L-methionine-dependent methyltransferases protein_coding superfamily protein;(source:Araport11) AT5G11960 AT5G11960.1 magnesium transporter, putative protein_coding (DUF803);(source:Araport11) AT4G00560 AT4G00560.4 NAD(P)-binding Rossmann-fold superfamily protein_coding protein;(source:Araport11) AT1G80510 AT1G80510.1 Encodes a close relative of the amino acid transporter ANT1 protein_coding (AT3G11900). AT2G21250 AT2G21250.1 NAD(P)-linked oxidoreductase superfamily protein_coding protein;(source:Araport11) AT5G04420 AT5G04420.1 Galactose oxidase/kelch repeat superfamily protein_coding protein;(source:Araport11) AT4G34910 AT4G34910.1 P-loop containing nucleoside triphosphate hydrolases protein_coding superfamily protein;(source:Araport11) AT5G66120 AT5G66120.2 3-dehydroquinate synthase;(source:Araport11) protein_coding AT1G45110 AT1G45110.1 Tetrapyrrole (Corrin/Porphyrin) protein_coding Methylase;(source:Araport11) AT1G67420 AT1G67420.2 Zn-dependent exopeptidases superfamily protein_coding protein;(source:Araport11) AT3G62370
    [Show full text]
  • Viewed and Published Immediately Upon Acceptance Cited in Pubmed and Archived on Pubmed Central Yours — You Keep the Copyright
    BMC Bioinformatics BioMed Central Methodology article Open Access Optimization based automated curation of metabolic reconstructions Vinay Satish Kumar1, Madhukar S Dasika2 and Costas D Maranas*2 Address: 1Department of Industrial and Manufacturing Engineering, The Pennsylvania State University, University Park, PA 16802, USA and 2Department of Chemical Engineering, The Pennsylvania State University, University Park, PA 16802, USA Email: Vinay Satish Kumar - [email protected]; Madhukar S Dasika - [email protected]; Costas D Maranas* - [email protected] * Corresponding author Published: 20 June 2007 Received: 14 December 2006 Accepted: 20 June 2007 BMC Bioinformatics 2007, 8:212 doi:10.1186/1471-2105-8-212 This article is available from: http://www.biomedcentral.com/1471-2105/8/212 © 2007 Satish Kumar et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Currently, there exists tens of different microbial and eukaryotic metabolic reconstructions (e.g., Escherichia coli, Saccharomyces cerevisiae, Bacillus subtilis) with many more under development. All of these reconstructions are inherently incomplete with some functionalities missing due to the lack of experimental and/or homology information. A key challenge in the automated generation of genome-scale reconstructions is the elucidation of these gaps and the subsequent generation of hypotheses to bridge them. Results: In this work, an optimization based procedure is proposed to identify and eliminate network gaps in these reconstructions. First we identify the metabolites in the metabolic network reconstruction which cannot be produced under any uptake conditions and subsequently we identify the reactions from a customized multi-organism database that restores the connectivity of these metabolites to the parent network using four mechanisms.
    [Show full text]