DOCSLIB.ORG

Mechanistic and Structural Studies of Salicylate Biosynthesis in Pseudomonas Aeruginosa

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Mechanistic and Structural Studies of Salicylate Biosynthesis in Pseudomonas Aeruginosa MECHANISTIC AND STRUCTURAL STUDIES OF SALICYLATE BIOSYNTHESIS IN PSEUDOMONAS AERUGINOSA BY Qianyi Luo Submitted to the Department of Molecular Biosciences and the Faculty of the Graduate School of the University of Kansas in partial fulfillment of the requirements for the degree of Doctor of Philosophy Chairperson – Audrey Lamb Committee members: Roberto De Guzman Krzysztof Kuczera Mark Richter Emily Scott William Picking Date defended: 4/22/2009 ii The Dissertation Committee for Qianyi Luo certifies that this is the approved version of the following dissertation: MECHANISTIC AND STRUCTURAL STUDIES OF SALICYLATE BIOSYNTHESIS IN PSEUDOMONAS AERUGINOSA Committee: Chairperson – Audrey Lamb Roberto De Guzman Krzysztof Kuczera Mark Richter Emily Scott William Picking Date approved: 4/23/2009 iii Abstract Iron is an essential element for most pathogenic bacteria. To survive and establish infections in host tissues, these pathogens must compete with the host organism for iron. One strategy is to excrete iron-chelator siderophores with very high affinity to ferric iron in the low iron environment of the host. The phenolate type siderophore, such as pyochelin in Pseudomonas aeruginosa, uses salicylate derived from chorismate as a precursor. Studies have shown that the salicylate activation by adenylation for incorporation to siderophores is associated with the growth and virulence of some pathogens. The inhibition of salicylate biosynthesis, and hence, siderophore production is considered an attractive target for the development of novel antimicrobial agents. In P. aeruginosa, salicylate is derived from chorismate via isochorismate by two enzymes: isochorismate synthase (PchA) and isochorismate-pyruvate lyase (IPL, PchB). PchB eliminates the enolpyruvyl side chain from isochorismate through a biologically unusual pericyclic reaction mechanism. PchB can also perform an adventitious pericyclic reaction that rearranges chorismate to prephenate possibly due to the homology to the E. coli chorismate mutase (CM). The primary contribution to lower the activation energy for enzymatic pericyclic reactions is controversial and may be arise from electrostatic stabilization of the transition state, or conformational stabilization of the reactive substrate. Structural and mutational studies on a key residue, lysine 42, of the active site loop suggest that rate enhancement of the two iv pericyclic reactions (IPL and CM) performed by PchB results from both the transition state stabilization and the reactive substrate conformation, but the relative contributions are different for each reaction. A mutation with less active site loop mobility, A43P indicates that the loop dynamics is related to catalysis. The I87T structure reveals a larger disordered region compared to the wild type structure, suggesting that conformational mobility may play a role in catalysis. PchA is an isochorismate synthase (ICS) in P. aeruginosa that removes the C4 hydroxyl group and adds a hydroxyl group to C2-chorismate. PchA is homologous to salicylate synthases from Yersinia spp. and Mycobacterium tuberculosis that convert chorismate to salicylate without requirement of an additional lyase such as PchB in P. aeruginosa. A sequence comparison between PchA with salicylate synthases of known structure suggests that two conserved residues are directly involved in the general acid and base chemistry in PchA: K221 as the general base and E269 as the general acid. Replacement of K221 and E269 with alanine respectively led to catalytically inactive enzymes, suggesting that K221 and E269 are critical for ICS catalysis. Preliminary pH dependence data for PchA supports the general acid and base mechanism of PchA catalysis. Two nonconserved residues A375 and D310 were also examined. Replacement of A375 by threonine, the corresponding residue in salicylate synthase, resulted in only residual ICS activity, indicating that A375 is not associated with the IPL-deficiency in PchA. The D310E mutant leads to two additional activities, IPL and CM. The additional activities in three reactions may due to the preferential orientation of substrates in the active site. v Acknowledgments I would like to thank everyone who helped me in my graduate education and research. I would like to acknowledge Dr. Richard H. Himes and Dr. Roberto De Guzman for their help with instrument setup for isochorismate purification. I would also like to acknowledge Dr. Richard L. Schowen for his enzymology lectures and his scientific advice in thermodynamic analyses. I am thankful to Dr. Kathy Meneely for her generous assistance in my graduate course work and my research. I would like to acknowledge all the undergraduates who helped with my projects: Krysti Spiess, Daniel Shippy, Tyler McMillen, Katie Waugh, and Allison Ho. Thank you to all of the previous and current members of the Lamb lab, Andy Haith, Dr. Lena Zaitseva, Jingping Lu, Andrew Ouellette, and Jose Olucha. I would like to thank Dr. T. Christopher Gamblin for his kind support and guidance with the instruments used in my research. I would like to thank the Protein Structure Laboratory at the University of Kansas for PchB K42A and I87T X-ray data collection. I would also like to thank my committee members for providing suggestions and reading this dissertation, especially Dr. Roberto De Guzman and Dr. Emily Scott who served as the readers. Finally, a very special thank you goes to my advisor, Dr. Audrey Lamb for her scientific expertise, significant guidance, continuous support, and encouragement throughout my graduate career. vi Table of Contents Abstract iii Acknowledgments v Table of Contents vi List of Figures viii List of Tables x CHAPTER 1 Introduction 1 1.1 Bacterial Infections 1 1.2 Bacterial Iron Uptake 2 The Importance of Iron 2 Heme as An Iron Source 4 Siderophores 6 1.3 Enzyme Active Site and Catalysis 16 Enzyme Active Site 16 Conformational Mobility in Enzyme 17 Transition State Theory for Enzyme Catalysis 18 1.4 Enzyme Catalytic Mechanisms 20 General Acid and Base Catalysis 20 Covalent Catalysis 21 Proximity and Orientation Effects 26 Preferential Binding of The Transition State 27 References 30 CHAPTER 2 Mechanistic and Structural Studies of PchB, a bifunctional Isochorismate-Pyruvate Lyase/Chorismate Mutase from Pseudomonas aeruginosa 34 2.1 Introduction 34 2.2 Materials and Methods 42 Site-Directed Mutagenesis of PchB 42 Overexpression and Purification of Wild Type and Mutant PchB 43 Circular Dichroism Studies 44 Protein Crystallization 44 Data Collection and Structure Determination 45 Isochorismate Preparation 47 Steady-State Kinetic Studies 48 Temperature Dependence of PchB Catalyzed Reactions 49 2.3 Results 50 Enzyme Production 50 CD Spectra Measurements 50 PchB K42A and I87T Structures 50 vii Isochorismate Preparation 55 Determination of Steady-State Kinetic Parameters 58 Determination of the Activation Parameters 60 2.4 Discussion 65 References 71 CHAPTER 3 Salicylate Synthetic Enzymes in Pseudomonas aeruginosa and Yersinia enterocolitica 75 3.1 Introduction 75 3.2 Material and Methods 85 3.2.1 Protein Production and Characterization 85 3.2.2 Steady-State Kinetic Studies 93 3.2.3 pH dependence of PchA catalysis 95 3.2.4 Protein Crystallization 96 3.3 Results and Discussion 99 CHAPTER 4 High Throughput Screening of Inhibitors of Salicylate Synthesis in Pseudomonas aeruginosa 123 4.1 Introduction 123 4.2 Materials and Methods 125 Protein Overproduction and Purification 125 Chorismate Preparation 125 HTS of Inhibitors of Coupled PchA and PchB Reaction 129 4.3 Results and Discussion 129 References 133 CHAPTER 5 Conclusion 134 References 138 viii List of Figures Figure Page 1-1 A schematic view of iron uptake in gram-negative bacteria 5 1-2 Representative siderophore structures 8 1-3 Representation of one cycle of the peptide chain elongation on a 10 module of NRPS 1-4 Pyochelin assembly line in P. aeruginosa 12 1-5 Yersiniabactin assembly line in Y. enterocolitica 14 1-6 Reaction diagram fro a single-substrate reaction in solution and 19 catalyzed by an enzyme 1-7 The transition states for ester hydrolysis catalyzed by a general 22 acid and a general base mechanisms 2-1 Reaction schemes of PchB catalyzed reactions 36 2-2 Structures of three chorismate mutases 37 2-3 Two near attack conformations (NACs) formed in PchB active site 41 2-4 Overlay of the circular dichroism spectra of wild type and mutant 51 PchB enzymes 2-5 PchB K42A structure 54 2-6 PchB I87T structure 56 2-7 Separation of isochorismate 57 2-8 Temperature dependence of kcat for chorismate mutase activity of 62 PchB 2-9 Temperature dependence of reaction rate for isochorismate 64 rearrangement to salicylate 3-1 Conversion of chorismate to isochorismate and its incorporation 76 into siderophores or menaquinone 3-2 MenF overall and active site structures 78 3-3 The general acid and base catalysis proposed by MenF 80 3-4 Salicylate synthase Irp9 81 3-5 Active site sequence alignment of five chorismate-utilizing 83 enzymes from MST family 3-6 Sequence alignment of PchA and Irp9 89 3-7 Superposition of wild type and mutants PchA elution profiles from 100 Superdex 200 gel filtration chromatography 3-8 Mg2+-dependence of PchA catalysis 103 3-9 The pH dependence of the PchA reaction rate 110 3-10 PchA buffer optimization screen 112 3-11 PchAt buffer optimization screen 113 3-12 PchA crystallized in sodium formate, pH 7.0 117 ix 3-13 A diffraction image of Irp9 118 4-1 1H-NMR spectrum of recrystallized chorismate 131 4-2 Coupled PchA and PchB assay using chorismate purified from 132
Load more

Recommended publications
  • Genomic Analyses of Unique Carbohydrate and Phytohormone Metabolism in the Macroalga Gracilariopsis Lemaneiformis (Rhodophyta)
    Sun et al. BMC Plant Biology (2018) 18:94 https://doi.org/10.1186/s12870-018-1309-2 RESEARCH ARTICLE Open Access Genomic analyses of unique carbohydrate and phytohormone metabolism in the macroalga Gracilariopsis lemaneiformis (Rhodophyta) Xue Sun1, Jun Wu2, Guangce Wang3, Yani Kang1,2, Hong Sain Ooi4, Tingting Shen2, Fangjun Wang1, Rui Yang1, Nianjun Xu1* and Xiaodong Zhao2* Abstract Background: Red algae are economically valuable for food and in industry. However, their genomic information is limited, and the genomic data of only a few species of red algae have been sequenced and deposited recently. In this study, we annotated a draft genome of the macroalga Gracilariopsis lemaneiformis (Gracilariales, Rhodophyta). Results: The entire 88.98 Mb genome of Gp. lemaneiformis 981 was generated from 13,825 scaffolds (≥500 bp) with an N50 length of 30,590 bp, accounting for approximately 91% of this algal genome. A total of 38.73 Mb of scaffold sequences were repetitive, and 9281 protein-coding genes were predicted. A phylogenomic analysis of 20 genomes revealed the relationship among the Chromalveolata, Rhodophyta, Chlorophyta and higher plants. Homology analysis indicated phylogenetic proximity between Gp. lemaneiformis and Chondrus crispus. The number of enzymes related to the metabolism of carbohydrates, including agar, glycoside hydrolases, glycosyltransferases, was abundant. In addition, signaling pathways associated with phytohormones such as auxin, salicylic acid and jasmonates are reported for the first time for this alga. Conclusion: We sequenced and analyzed a draft genome of the red alga Gp. lemaneiformis, and revealed its carbohydrate metabolism and phytohormone signaling characteristics. This work will be helpful in research on the functional and comparative genomics of the order Gracilariales and will enrich the genomic information on marine algae.
    [Show full text]
  • Letters to Nature
    letters to nature Received 7 July; accepted 21 September 1998. 26. Tronrud, D. E. Conjugate-direction minimization: an improved method for the re®nement of macromolecules. Acta Crystallogr. A 48, 912±916 (1992). 1. Dalbey, R. E., Lively, M. O., Bron, S. & van Dijl, J. M. The chemistry and enzymology of the type 1 27. Wolfe, P. B., Wickner, W. & Goodman, J. M. Sequence of the leader peptidase gene of Escherichia coli signal peptidases. Protein Sci. 6, 1129±1138 (1997). and the orientation of leader peptidase in the bacterial envelope. J. Biol. Chem. 258, 12073±12080 2. Kuo, D. W. et al. Escherichia coli leader peptidase: production of an active form lacking a requirement (1983). for detergent and development of peptide substrates. Arch. Biochem. Biophys. 303, 274±280 (1993). 28. Kraulis, P.G. Molscript: a program to produce both detailed and schematic plots of protein structures. 3. Tschantz, W. R. et al. Characterization of a soluble, catalytically active form of Escherichia coli leader J. Appl. Crystallogr. 24, 946±950 (1991). peptidase: requirement of detergent or phospholipid for optimal activity. Biochemistry 34, 3935±3941 29. Nicholls, A., Sharp, K. A. & Honig, B. Protein folding and association: insights from the interfacial and (1995). the thermodynamic properties of hydrocarbons. Proteins Struct. Funct. Genet. 11, 281±296 (1991). 4. Allsop, A. E. et al.inAnti-Infectives, Recent Advances in Chemistry and Structure-Activity Relationships 30. Meritt, E. A. & Bacon, D. J. Raster3D: photorealistic molecular graphics. Methods Enzymol. 277, 505± (eds Bently, P. H. & O'Hanlon, P. J.) 61±72 (R. Soc. Chem., Cambridge, 1997).
    [Show full text]
  • Exploring the Chemistry and Evolution of the Isomerases
    Exploring the chemistry and evolution of the isomerases Sergio Martínez Cuestaa, Syed Asad Rahmana, and Janet M. Thorntona,1 aEuropean Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, United Kingdom Edited by Gregory A. Petsko, Weill Cornell Medical College, New York, NY, and approved January 12, 2016 (received for review May 14, 2015) Isomerization reactions are fundamental in biology, and isomers identifier serves as a bridge between biochemical data and ge- usually differ in their biological role and pharmacological effects. nomic sequences allowing the assignment of enzymatic activity to In this study, we have cataloged the isomerization reactions known genes and proteins in the functional annotation of genomes. to occur in biology using a combination of manual and computa- Isomerases represent one of the six EC classes and are subdivided tional approaches. This method provides a robust basis for compar- into six subclasses, 17 sub-subclasses, and 245 EC numbers cor- A ison and clustering of the reactions into classes. Comparing our responding to around 300 biochemical reactions (Fig. 1 ). results with the Enzyme Commission (EC) classification, the standard Although the catalytic mechanisms of isomerases have already approach to represent enzyme function on the basis of the overall been partially investigated (3, 12, 13), with the flood of new data, an integrated overview of the chemistry of isomerization in bi- chemistry of the catalyzed reaction, expands our understanding of ology is timely. This study combines manual examination of the the biochemistry of isomerization. The grouping of reactions in- chemistry and structures of isomerases with recent developments volving stereoisomerism is straightforward with two distinct types cis-trans in the automatic search and comparison of reactions.
    [Show full text]
  • Physcomitrium Patens Infection by Colletotrichum Gloeosporioides: Understanding the Fungal–Bryophyte Interaction by Microscopy, Phenomics and RNA Sequencing
    Journal of Fungi Article Physcomitrium patens Infection by Colletotrichum gloeosporioides: Understanding the Fungal–Bryophyte Interaction by Microscopy, Phenomics and RNA Sequencing Adriana Otero-Blanca 1 , Yordanis Pérez-Llano 1 , Guillermo Reboledo-Blanco 2, Verónica Lira-Ruan 1 , Daniel Padilla-Chacon 3, Jorge Luis Folch-Mallol 4 , María del Rayo Sánchez-Carbente 4 , Inés Ponce De León 2 and Ramón Alberto Batista-García 1,* 1 Centro de Investigación en Dinámica Celular, Instituto de Investigación en Ciencias Básicas y Aplicadas, Universidad Autónoma del Estado de Morelos, Cuernavaca 62209, Mexico; [email protected] (A.O.-B.); [email protected] (Y.P.-L.); [email protected] (V.L.-R.) 2 Departamento de Biología Molecular, Instituto de Investigaciones Biológicas Clemente Estable, Montevideo 11600, Uruguay; [email protected] (G.R.-B.); [email protected] (I.P.D.L.) 3 Consejo Nacional de Ciencia y Tecnología (CONACyT), Colegio de Postgraduados de México, Campus Montecillo, Texcoco 56230, Mexico; [email protected] 4 Centro de Investigación en Biotecnología, Universidad Autónoma del Estado de Morelos, Cuernavaca 62209, Mexico; [email protected] (J.L.F.-M.); [email protected] (M.d.R.S.-C.) Citation: Otero-Blanca, A.; * Correspondence: [email protected] or [email protected]; Tel.: +52-777-3297020 Pérez-Llano, Y.; Reboledo-Blanco, G.; Lira-Ruan, V.; Padilla-Chacon, D.; Abstract: Anthracnose caused by the hemibiotroph fungus Colletotrichum gloeosporioides is a dev- Folch-Mallol, J.L.; Sánchez-Carbente, astating plant disease with an extensive impact on plant productivity. The process of colonization M.d.R.; Ponce De León, I.; and disease progression of C.
    [Show full text]
  • Structure of Isochorismate Synthase in Complex with Magnesium
    short communications Acta Crystallographica Section D Biological Structure of isochorismate synthase in complex Crystallography with magnesium ISSN 0907-4449 James F. Parsons,a* Katherine M. The electron carrier menaquinone is one of many important bacterial Shia and Jane E. Ladnera,b metabolites that are derived from the key intermediate chorismic acid. MenF, the first enzyme in the menaquinone pathway, catalyzes the isomerization of chorismate to isochorismate. Here, an improved structure of MenF in a new a Center for Advanced Research in crystal form is presented. The structure, solved at 2.0 A˚ resolution in complex Biotechnology, University of Maryland Biotechnology Institute, USA, and bNational with magnesium, reveals a well defined closed active site. Existing evidence Institute of Standards and Technology, USA suggests that the mechanism of the reaction catalyzed by MenF involves nucleophilic attack of a water molecule on the chorismate ring. The structure reveals a well defined water molecule located in an appropriate position for Correspondence e-mail: activation by Lys190 and attack on the substrate. [email protected] Received 23 January 2008 Accepted 26 February 2008 1. Introduction The menaquinone-specific isochorismate synthase (MenF) from Escherichia coli is a chorismate-utilizing enzyme that catalyzes the formation of isochorismate for use in menaquinone biosynthesis PDB References: apo-MenF, 3bzm, r3bzmsf; MenF–Mg2+ complex, 3bzn, r3bznsf. (Fig. 1; Daruwala et al., 1997). Menaquinone is an electron carrier that is essential for anaerobic electron transport in bacteria. Isochorismate is also an intermediate in the biosynthesis of sidero- phores such as the iron chelator enterobactin. The E. coli genome encodes a second isochorismate synthase, EntC, which is involved in this pathway (Buss et al., 2001).
    [Show full text]
  • Iversidade Técnica De Lisboa Instituto Superior De Agronomia
    UNIVERSIDADE TÉCNICA DE LISBOA INSTITUTO SUPERIOR DE AGRONOMIA Alternative strategies to fight apple scab Mariana da Silva Gomes Mota ORIENTADOR: Doutora Cristina Maria Moniz Simões Oliveira CO-ORIENTADOR: Doutora Margit Laimer JÚRI: Presidente: Reitor da Universidade Técnica de Lisboa Vogais: Doutora Joana Maria Canelhas Palminha Duclos, professora catedrática do Instituto Superior de Agronomia da Universidade Técnica de Lisboa; Doutora Margit Laimer, professora do Institute of Applied Microbiology da University of Agriculture, Viena, Áustria; Doutora Cristina Maria Moniz Simões Oliveira, professora associada do Instituto Superior de Agronomia da Universidade Técnica de Lisboa; Doutora Maria Luísa Lopes de Castro e Brito, professora auxiliar do Instituto Superior de Agronomia da Universidade Técnica de Lisboa; Doutora Maria Antonieta Piçarra Pereira, professora Adjunta da Escola Superior Agrária de Castelo Branco; Doutora Helene Maria Pühringer, investigadora do Institute of Applied Microbiology da University of Agriculture, Viena, Áustria, na qualidade de especialista. Doutoramento em Engenharia Agronómica Lisboa 2002 UNIVERSIDADE TÉCNICA DE LISBOA INSTITUTO SUPERIOR DE AGRONOMIA Alternative strategies to fight apple scab Mariana da Silva Gomes Mota ORIENTADOR: Doutora Cristina Maria Moniz Simões Oliveira CO-ORIENTADOR: Doutora Margit Laimer JÚRI: Presidente: Reitor da Universidade Técnica de Lisboa Vogais: Doutora Joana Maria Canelhas Palminha Duclos, professora catedrática do Instituto Superior de Agronomia da Universidade Técnica
    [Show full text]
  • Syntheses of Chorismate Analogs for Investigation of Structural Requirements for Chorismate Mutase
    Syntheses of Chorismate Analogs for Investigation of Structural Requirements for Chorismate Mutase by Chang-Chung Cheng B.S., Department of Chemistry National Chung-Hsing University 1986 Submitted to the Department of Chemistry in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Organic Chemistry at the Massachusetts Institute of Technology May, 1994 © 1994 Chang-Chung Cheng All rights reserved The author hereby grants to MIT permission to reproduce and to distribute publicly paper and electronic copies of this thesis document in whole or in oart. Signature of Author... ............ ............ ......................................................... Department of Chemistry May 18, 1994 Certified by...................................... Glenn A. Berchtold Thesis Supervisor Accepted by..........................................-. ,..... ......................................................... Glenn A. Berchtold Science Chairman, Departmental Committee on Graduate Studies JUN 2 1 1994 LjENrVUVk*_ This doctoral thesis has been examined by a Committee of the Department of Chemistry as Follows: Professor Frederick D. Greene, II......... .......... Chairman Professor Glenn A. Berchtold .ThS................................ Thesis Supervisor Professor Julius Rebek, Jr............... .. ............................................L% 1 Foreword Chang-Chung (Cliff) Cheng's Ph. D. thesis is a special thesis for me because of our association with the individual who supervised his undergraduate research and since Cliff will be the last student to receive the Ph. D. from me during my career at M.I.T.. Cliff received the B.S. degree in Chemistry from National Chung-Hsing University, Taichung, Taiwan in June 1986, and he enrolled in our doctoral program in September 1989. During his undergraduate career Cliff was engaged in research under the supervision of Professor Teng-Kuei Yang. Professor Yang also received his undergraduate training in chemistry at National Chung-Hsing University before coming to the United States to complete the Ph.
    [Show full text]
  • Building Microbial Factories for The
    Contents lists available at ScienceDirect Metabolic Engineering journal homepage: www.elsevier.com/locate/meteng Building microbial factories for the production of aromatic amino acid pathway derivatives: From commodity chemicals to plant-sourced natural products ∗ Mingfeng Caoa,b,1, Meirong Gaoa,b,1, Miguel Suásteguia,b, Yanzhen Meie, Zengyi Shaoa,b,c,d, a Department of Chemical and Biological Engineering, Iowa State University, USA b NSF Engineering Research Center for Biorenewable Chemicals (CBiRC), Iowa State University, USA c Interdepartmental Microbiology Program, Iowa State University, USA d The Ames Laboratory, USA e School of Life Sciences, No.1 Wenyuan Road, Nanjing Normal University, Qixia District, Nanjing, 210023, China ARTICLE INFO ABSTRACT Keywords: The aromatic amino acid biosynthesis pathway, together with its downstream branches, represents one of the Aromatic amino acid biosynthesis most commercially valuable biosynthetic pathways, producing a diverse range of complex molecules with many Shikimate pathway useful bioactive properties. Aromatic compounds are crucial components for major commercial segments, from De novo biosynthesis polymers to foods, nutraceuticals, and pharmaceuticals, and the demand for such products has been projected to Microbial production continue to increase at national and global levels. Compared to direct plant extraction and chemical synthesis, Flavonoids microbial production holds promise not only for much shorter cultivation periods and robustly higher yields, but Stilbenoids Benzylisoquinoline alkaloids also for enabling further derivatization to improve compound efficacy by tailoring new enzymatic steps. This review summarizes the biosynthetic pathways for a large repertoire of commercially valuable products that are derived from the aromatic amino acid biosynthesis pathway, and it highlights both generic strategies and spe- cific solutions to overcome certain unique problems to enhance the productivities of microbial hosts.
    [Show full text]
  • Supplemental Table S1: Comparison of the Deleted Genes in the Genome-Reduced Strains
    Supplemental Table S1: Comparison of the deleted genes in the genome-reduced strains Legend 1 Locus tag according to the reference genome sequence of B. subtilis 168 (NC_000964) Genes highlighted in blue have been deleted from the respective strains Genes highlighted in green have been inserted into the indicated strain, they are present in all following strains Regions highlighted in red could not be deleted as a unit Regions highlighted in orange were not deleted in the genome-reduced strains since their deletion resulted in severe growth defects Gene BSU_number 1 Function ∆6 IIG-Bs27-47-24 PG10 PS38 dnaA BSU00010 replication initiation protein dnaN BSU00020 DNA polymerase III (beta subunit), beta clamp yaaA BSU00030 unknown recF BSU00040 repair, recombination remB BSU00050 involved in the activation of biofilm matrix biosynthetic operons gyrB BSU00060 DNA-Gyrase (subunit B) gyrA BSU00070 DNA-Gyrase (subunit A) rrnO-16S- trnO-Ala- trnO-Ile- rrnO-23S- rrnO-5S yaaC BSU00080 unknown guaB BSU00090 IMP dehydrogenase dacA BSU00100 penicillin-binding protein 5*, D-alanyl-D-alanine carboxypeptidase pdxS BSU00110 pyridoxal-5'-phosphate synthase (synthase domain) pdxT BSU00120 pyridoxal-5'-phosphate synthase (glutaminase domain) serS BSU00130 seryl-tRNA-synthetase trnSL-Ser1 dck BSU00140 deoxyadenosin/deoxycytidine kinase dgk BSU00150 deoxyguanosine kinase yaaH BSU00160 general stress protein, survival of ethanol stress, SafA-dependent spore coat yaaI BSU00170 general stress protein, similar to isochorismatase yaaJ BSU00180 tRNA specific adenosine
    [Show full text]
  • Salicylic Acid Biosynthesis in Plants
    fpls-11-00338 April 16, 2020 Time: 18:0 # 1 MINI REVIEW published: 17 April 2020 doi: 10.3389/fpls.2020.00338 Salicylic Acid Biosynthesis in Plants Hannes Lefevere, Lander Bauters and Godelieve Gheysen* Department of Biotechnology, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium Salicylic acid (SA) is an important plant hormone that is best known for mediating host responses upon pathogen infection. Its role in plant defense activation is well established, but its biosynthesis in plants is not fully understood. SA is considered to be derived from two possible pathways; the ICS and PAL pathway, both starting from chorismate. The importance of both pathways for biosynthesis differs between plant species, rendering it hard to make generalizations about SA production that cover the entire plant kingdom. Yet, understanding SA biosynthesis is important to gain insight into how plant pathogen responses function and how pathogens can interfere with them. In this review, we have taken a closer look at how SA is synthesized and the importance of both biosynthesis pathways in different plant species. Keywords: salicylic acid biosynthesis, isochorismate synthase, phenylalanine ammonia-lyase, plant defense, Edited by: pathogen infection Danièle Werck, CNRS, UPR2357 Institut de Biologie Moléculaire des Plantes (IBMP), France INTRODUCTION Reviewed by: Salicylic acid (SA) was reported to play a role in disease resistance in tobacco plants by White Thierry Heitz, already in 1979 (White, 1979). Since then, the importance of SA in plant defense to biotic and UPR2357 Institut de Biologie Moléculaire des Plantes (IBMP), abiotic stimuli has been well established. SA levels are known to increase in many pathosystems France upon infection with viruses, fungi, insects, and bacteria (Ogawa et al., 2006; Kim and Hwang, 2014; Jürgen Zeier, Hao et al., 2018; Zhao et al., 2019), and exogenous SA treatment boosts the defense system of the Heinrich Heine University Düsseldorf, host (Nahar et al., 2011; Wang and Liu, 2012; Tripathi et al., 2019; Zhang and Li, 2019).
    [Show full text]
  • POLSKIE TOWARZYSTWO BIOCHEMICZNE Postępy Biochemii
    POLSKIE TOWARZYSTWO BIOCHEMICZNE Postępy Biochemii http://rcin.org.pl WSKAZÓWKI DLA AUTORÓW Kwartalnik „Postępy Biochemii” publikuje artykuły monograficzne omawiające wąskie tematy, oraz artykuły przeglądowe referujące szersze zagadnienia z biochemii i nauk pokrewnych. Artykuły pierwszego typu winny w sposób syntetyczny omawiać wybrany temat na podstawie możliwie pełnego piśmiennictwa z kilku ostatnich lat, a artykuły drugiego typu na podstawie piśmiennictwa z ostatnich dwu lat. Objętość takich artykułów nie powinna przekraczać 25 stron maszynopisu (nie licząc ilustracji i piśmiennictwa). Kwartalnik publikuje także artykuły typu minireviews, do 10 stron maszynopisu, z dziedziny zainteresowań autora, opracowane na podstawie najnow­ szego piśmiennictwa, wystarczającego dla zilustrowania problemu. Ponadto kwartalnik publikuje krótkie noty, do 5 stron maszynopisu, informujące o nowych, interesujących osiągnięciach biochemii i nauk pokrewnych, oraz noty przybliżające historię badań w zakresie różnych dziedzin biochemii. Przekazanie artykułu do Redakcji jest równoznaczne z oświadczeniem, że nadesłana praca nie była i nie będzie publikowana w innym czasopiśmie, jeżeli zostanie ogłoszona w „Postępach Biochemii”. Autorzy artykułu odpowiadają za prawidłowość i ścisłość podanych informacji. Autorów obowiązuje korekta autorska. Koszty zmian tekstu w korekcie (poza poprawieniem błędów drukarskich) ponoszą autorzy. Artykuły honoruje się według obowiązujących stawek. Autorzy otrzymują bezpłatnie 25 odbitek swego artykułu; zamówienia na dodatkowe odbitki (płatne) należy zgłosić pisemnie odsyłając pracę po korekcie autorskiej. Redakcja prosi autorów o przestrzeganie następujących wskazówek: Forma maszynopisu: maszynopis pracy i wszelkie załączniki należy nadsyłać w dwu egzem­ plarzach. Maszynopis powinien być napisany jednostronnie, z podwójną interlinią, z marginesem ok. 4 cm po lewej i ok. 1 cm po prawej stronie; nie może zawierać więcej niż 60 znaków w jednym wierszu nie więcej niż 30 wierszy na stronie zgodnie z Normą Polską.
    [Show full text]
  • Xnal $750. Bam H
    US 2011 0099668A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2011/0099668A1 Singh et al. (43) Pub. Date: Apr. 28, 2011 (54) EXPRESSING GLKN PLANTS Publication Classification (51) Int. Cl. Inventors: Jasbir Singh, Ottawa (CA); AOIH 5/00 (2006.01) (76) CI2N 5/82 (2006.01) Ghislaine C. Allard, Val des Monts CI2N 15/63 (2006.01) (CA); Leonid V. Savitch, Kanata AOIH I/00 (2006.01) (CA); Rajagopal Subramaniam, CI2N 5/04 (2006.01) Ottawa (CA) C7H 2L/04 (2006.01) (52) U.S. C. ... 800/279; 536/23.1; 800/301; 435/320.1; (21) Appl. No.: 12/151,046 435/419:435/412: 435/.414; 435/417; 435/416; 800/278; 800/298 (22) Filed: May 1, 2008 (57) ABSTRACT The present invention provides, in part, GLK1 nucleic acid Related U.S. Application Data molecules and polypeptides that can be used to confer resis tance to a pathogen in a plant. The present invention also (60) Provisional application No. 60/915,294, filed on May provides methods of detecting disease resistance genes and 1, 2007. plants. Pyu I (8945) EcoRI(876), SacI(8764) Snalt 8752) : xnal $750. Bam H. (8745) 1. Ahal (8739), W Scal (4) SalI (8733). 1 Scal (14) Psi (8731), \ | Scal (24) HindIII (8715), . Scal(34) Psil (8707) 2x P35s NotI(882) EcoRV (8045) XhoI (7948) s \,w \ EW onhancern bar oRF --- Sali (7534). Pyuli (7307) - Koni (7289) pTF101.1 Tvsp - Notl (2415) PyuII (6596) 25 (64.27). LB ClaI(3037) Bell (6190) Bel(5703) ApaL (5626) / Noti (3705) aad A ApaL (4532) ApaL (4034) pBR322 Patent Application Publication Apr.
    [Show full text]