Genetics of siderophore assembly line biosynthesis in Vibrio anguillarum. by Manuela Di Lorenzo A DISSERTATION Presented to the Department of Molecular Microbiology and Immunology and the Oregon Health and Science University School of Medicine in partial fulfillment of the requirements for the degree of Doctor of Philosophy June 2005 School of Medicine Oregon Health Sciences University Department of Molecular Microbiology & Immunology CERTIFICATE OF APPROVAL This is certify that the Ph.D. thesis of Manuela Di Lorenzo has been approved Jo . Crosa, P .D., Professor in charge of thesis Mathew Sachs, Ph.D. David F ell, Ph.D. TABLE OF CONTENTS Table of contents Table of abbreviations Vll Acknowledgements XIV Abstract XV Chapter 1 1.0. Introduction 2 1.1. Bacterial iron acquisition systems 5 1.1.1. Utilization of iron proteins of the host 7 1.1.2. Siderophore-mediated iron uptake systems 10 1.2. Nonribosomal peptide synthetases and siderophore synthesis 15 1. 3. Regulation of bacterial iron uptake systems 21 1.4. Vibrio anguillarum iron acquisition systems 24 1.5. Virulence plasmids and iron acquisition systems 32 1.6. Summary of the work presented in this thesis 34 Chapter 2 62 2.0. Abstract 63 2.1. Introduction 64 2.2. Materials and methods 66 2.2.1. Bacterial strains, plasmids, and growth conditions 66 2.2.2. Determination of the pJM 1 sequence 66 2.2.3. Nucleotide sequences accession number 67 2.3. Results 68 2.3.1. Nucleotide composition 68 2.3.2. ORF analysis 68 2.3.3. Utilization of iron 69 2.3.4. Insertion elements and transposon 74 2.3.5. Replication and partition 75 2.3.6. The microheterogeneity of pJM 1-like plasmids in strains of V anguillarum 76 2.4. Discussion 77 2.5. Acknowledgements 81 Chapter 3 91 3.0. Abstract 92 3.1. Introduction 93 3.2. Materials and methods 95 3.2.1. Bacterial strains, plasmids, and growth conditions 95 3.2.2. General methods 95 .., 7 .., .).-. .). Construction of the complementing clone 96 3.2.4. Site-directed mutagenesis 97 3.2.5. Construction of V anguillarum mutant strains by allelic-exchange 98 3.2.6. Determination of DHBA and anguibactin production 99 3.2.7. AngBp protein expression and purification 99 3.2.8. Expression and purification of EntC, Sfp and Vi bE 100 11 3.2.9. Assay for isochorismate lyase activity 101 3.2.1 0. Analysis of covalent [14C] salicylation of the ArCP domain 101 3 .2 .11. Nucleotide sequences accession number 102 3.3. Results 103 3.3.1. Identification of a chromosomal angB gene in strain 775 103 3.3.2. Mutational analysis of the angBp gene in strain 775 104 3.3.3. Mutational analysis of the angBc gene in strain 775 and generation of a double angB mutant in strain 775 105 3.3 .4. Characterization of the AngB functions in vivo 106 3.3.5. ICL and ArCP domain activities in vitro 108 3.4. Discussion 110 3. 5. Acknowledgements 113 Chapter 4 127 4.0. Abstract 128 4.1. Introduction 129 4.2. Materials and methods 131 4.2.1. Bacterial strains, plasmids, and growth conditions 131 4.2.2. General methods 131 4.2.3. Construction ofthe complementing clone 132 4.2.4. Site-directed mutagenesis 133 4.2.5. Construction of promoter fusions in pKK232-8 133 Ill 4.2.6. Construction of V anguillarum strains by conjugation and allelic-exchange 134 4.2.7. Growth in iron-limiting conditions and detection of anguibactin 134 4.2.8. Fish infectivity assays 135 4.2.9. CAT assay 135 4.2.10. RNA isolation 135 4.2.11. Primer extension 136 4.2.12. RNase protection assay 136 4.3. Results 138 4.3.1. Sequencing and analysis of a transposon insertion in the angM gene 138 4.3.2. Construction of an angM mutant in plasmid pJM 1 and complementation with the cloned angM gene 138 4.3.3. PCP and C domains of AngM are essential for anguibactin biosynthesis 140 4.3.4. Effect of angM mutations on the virulence phenotype of V anguillarum 775 141 4.3.5. Transcription and regulation of the angM gene 141 4.4. Discussion 144 4.5. Acknowledgements 148 Chapter 5 165 5.0. Abstract 166 IV 5.1. Introduction 167 5.2. Materials and methods 169 5.2.1. Bacterial strains and plasmids 169 5.2.2. General methods 169 5.2.3. Mobilization of the # 120 insertion mutant in angN from pJHC-T2612 to the pJM1 plasmid 170 5 .2.4. Construction of the complementing clone 170 5.2.5. Site-directed mutagenesis 171 5.2.6. Growth in iron-limiting conditions and detection of anguibactin 171 5.2.7. Fish infectivity assays 172 5.2.8. RNA isolation 172 5.2.9. Primer extension 173 5.2.10. Ribonuclease protection assays 173 5.3. Results 175 5.3.1. Analysis of the AngN sequence 175 5.3.2. Disruption of the angN gene and complementation with a wild type angN gene 175 5.3.3. Effect of site-directed modification of the angN gene on anguibactin production 176 5.3.4. AngN cyclization domains and the virulence of V anguillarum 178 5.3.5. Transcription and regulation of the angN gene 178 v 5.4. Discussion 181 5.5. Acknowledgements 184 Chapter 6 197 6.0. Discussion 198 6.1. Summary and conclusions 207 References 210 Vl TABLE OF ABBREVIATIONS A (DNA context) deoxyadenosine A (protein context) alanine A (NRPS domain) adenylation a alpha ABC A TP binding cassette AcCoA acetyl coenzyme A ACP acyl carrier protein ADP adenosine diphosphate Ap ampicillin ArCP aryl carrier protein ATP adenosine triphosphate ATPase adenosine triphosphatase ~ beta bp base pairs BSA bovine serum albumin C (DNA context) deoxycytosine C (protein context) cysteine C (NRPS domain) condensation C- carboxy- oc degree Celsius CAS chrome azurol S VII CAT chloramphenicol acetyl transferase Cl chloride Cm chloramphenicol Co A coenzyme A cpm counts per minute Cy cyclization D aspartic acid Da dalton DHB 2,3-dihydroxybenzoy I DHBA 2,3-dihydroxybenzoic acid DHBS 2,3-dihydroxybenzoylserine DHP dihydroxyphenyl DHPT dihydroxyphenylthiazolyl DNA deoxyribonucleic acid DNase deoxyribonuclease OTT dithiothreitol E (protein context) glutamic acid E (NRPS domain) epimerization extinction coefficient ECF extracytoplasmic function EDDA ethy lenediamine-di -( o­ hydroxyphenyl acetic acid) EDTA ethylenediaminetetraacetate Vlll E2p dihydrolipoyl transacetylases F pheny !alanine FAD flavin adenine dinucleotide Fe Iron ferrous iron Fe 3+ ferric iron G (DNA context) deoxyguanosine G (protein context) glycine g gram y gamma Ga gallium Gm gentamycin H (protein context) histidine H (chemical compounds) hydrogen h hour isoleucine ICL isochorismate lyase IPTG isopropyl-13-D-thiogalactoside IS insertion sequence ITB iron transport biosynthesis K (protein context) lysine K (chemical compounds) potassium kb kilobase pairs IX kDa kilo Dalton Km kanamycin L leucine A. lambda LB Luria-Bertani medium LDso 50% lethal dose LDH L-lactate dehydrogenase M (protein context) methionine M molar Mg magnesium mg milligram !-tg microgram MIC minimal inhibitory concentration mm minute ml milliliter !-!1 microliter mM millimolar !-!M micro molar mOX methyloxazolinyl mRNA messenger RNA MT methylation N aspa!agme N- ammo- X Na sodium NADH nicotinamide adenine dinucleotide NADPH nicotinamide adenine dinucleotide phosphate ng nanogram nM nanomolar nm nanometer nmol nanomole NRPS nonribosomal peptide synthetase NSPD norspermidine 0 oxygen ODx optical density at x nm OMR outer membrane receptor ORF open reading frame Ox oxidase p proline PAGE polyacrylamide gel electrophoresis PBP periplasm binding protein PCP peptidyl carrier protein PCR polymerase chain reaction % percent piTBO promoter of the iron transport biosynthesis operon Xl PMF proton motive force pp phosphopantetheiny 1 Q glutamine R argmme Red reductase Rif rifampicin RNA ribonucleic acid RNase ribonuclease s senne s seconds SDS sodium dodecyl sulfate Sp spectinomycin T (protein context) threonine T (DNA context) deoxythymidine TAF transacting factor Tc tetracycline TCA trichloroacetic acid TCEP tris( carboxyethy I)phosphine TE thioesterase Tp trimethropim TSBS trypticase soy broth with 1% NaCl TSAS trypticase soy agar with 1% NaCl UTP uridine triphosphate Xll v valine w tryptophan y tyrosine Zn zmc Xlll Acknowledgements. I would like to thank Jorge and Lidia Crosa for ail their support and the way they always made me feel at home. Also many thanks to Marcelo Tolmasky and Luis Actis for the fruitful and fun discussions on my thesis project but also on several non-scientific subjects. To Kirsten Mattison and the past and present members of the lab, I owe gratitude for the help they gave me in the progress of this work. I am also grateful to the members of my committee for their support in the development of my project. I could have not done much of the work for this thesis without the assistance of Jeff Vandehey and Chris Langford during my many computer crises. I also want to thank my parents: Grazie per aver condiviso ogni mia ambizione e per l'aiuto che mi avete sempre dato, anche in questa avventura in terra straniera. Finally, I am very glad for the presence in my work and private life of Michie! Stork that always supports me and helps me overcoming any obstacle. XlV Abstract The pathogen Vibrio anguillarum causes a fatal hemorrhagic septicemia in salmonid fishes and many isolates of this bacterium possess a plasmid-mediated iron uptake system that has been shown to be essential for virulence. This iron acquisition system centers on the siderophore anguibactin that is synthesized from 2,3- dihydroxybenzoic acid, cysteine and histidine via a nonribosomal peptide synthetase mechanism. Nonribosomal peptide synthetases (NRPSs) are multimodular proteins that assemble peptides in the absence of an RNA template.