Identification and Characterisation of Novel Iron Acquisition Mechanisms in Sinorhizobium

Declaration

I hereby certify that this material, which I now submit for assessment on the programme of study leading to the award of Degree of Doctor of Philosophy is entirely my own work, that I have exercised reasonable care to ensure that the work is original, and does not to the best of my knowledge breach any law of copyright, and has not been taken from the work of others save and to the extent that such work has been cited and acknowledged within the text of my own work.

Signed: ______ID No.: 50380741 Damien Keogh Date: 27th May 2008

I Acknowledgements

The work presented in this thesis is the fruition of years of work with support from colleagues, friends and family…..

I would particularly like to thank Dr. Michael O’Connell for giving me the opportunity to pursue a Ph.D. in his research group and for his guidance and encouragement over the years. Within Dr. O’Connell’s research group, two postdoctoral researchers stood out as scientific role models for me, my friend and lab mentor Dr. Páraic Ó Cuív whose knowledge and expertise were an invaluable resource and Dr. Paul Clarke whose encouragement and advice was much appreciated. I also wish to acknowledge the support of the postdocs, postgrads and technicians in the DCU School of Biotechnology and the visiting summer researchers to the lab that worked with me over the years.

Also, I would like to express my appreciation to the group of Prof. Peter J.F. Henderson in the School of Biochemistry and Molecular Biology at the University of Leeds for being so accommodating during my research visit and for their help with the project. In particular, I would like to thank Pikyee Mia and Dr. Nicholas G. Rutherford for their friendship and technical support during my stay.

It would not have been possible to complete my thesis without my friends and family. Mom and Dad, you have worked so hard to allow me take advantage of this fantastic opportunity.....this thesis is for you.

II Publications

Papers

Ó Cuív, P., Keogh, D., Clarke, P., and O’Connell, M. (2008). “The hmuUV genes of Sinorhizobium meliloti 2011 encode the permease and ATPase components of an ABC transport system for the utilisation of both haem and hydroxamate siderophores, ferrichrome and ferrioxamine B” Accepted for publication in Molecular Microbiology subject to revision of the manuscript.

Ó Cuív, P., Keogh, D., Clarke, P., and O’Connell, M. (2007). “FoxB of Pseudomonas aeruginosa functions in the utilisation of the xenosiderophores ferrichrome, ferrioxamine B, and schizokinen: Evidence for transport redundancy at the inner membrane”. J Bacteriol 189(1): 284-287.

Keogh, D., Ó Cuív, P., Clarke, P., Mia, P., Rutherford, N.G., Henderson, P.J.F., and O’Connell, M. “Interaction of the xenosiderophore ferrichrome with the novel single unit siderophore transporter FoxB of Pseudomonas aeruginosa” Manuscript in preparation for the Journal of Analytical Biochemistry.

Keogh, D., Ó Cuív, P., Clarke, P., and O’Connell, M. “Hydroxamate siderophore mediated iron acquisition by the permease and ATPase components of haem transport systems” Manuscript in preparation for the Journal of Microbiology.

Presentations

“ Novel Bacterial Iron Transporters of the MFS and ABC Superfamilies”. Society for General Microbiology, Young Microbiologist of the Year Final, The University of Edinburgh, September 2007.

“Splitting the Model: Novel ABC Iron Transport Systems”. School of Biotechnology, Biotechnology Seminar Series, Dublin City University, Ireland, April 2007.

III Posters

Keogh, D., Ó Cuív, P., Clarke, P., and O’Connell, M. (2007). “Transport of hydroxamate siderophores in Sinorhizobium meliloti” Society for General Microbiology 160th Meeting, University of Manchester.

Ó Cuív, P., Keogh, D., Clarke, P., and O’Connell, M. (2006). “Sinorhizobium meliloti and Pseudomonas aeruginosa encode novel transport systems for the inner membrane transport of hydroxamate siderophores” 5th International Biometals Symposium, Oregon USA.

Ó Cuív, P., Keogh, D., Clarke, P., and O’Connell, M. (2006). “Molecular characterisation of citrate hydroxamate and hydroxamate type siderophore acquisition systems by Pseudomonas aeruginosa” Society for General Microbiology, Irish Branch Symposium, Trinity College Dublin.

Ó Cuív, P., Keogh, D., Clarke, P., and O’Connell, M. (2005). “Novel mechanisms for the transport of hydroxamate siderophores” 7th International Symposium on Microbial Iron Transport Storage and Metabolism, Institut Pasteur, Paris France.

Viguier,C., Ó Cuív, P., Keogh, D., Clarke, P., and O’Connell, M. (2005). “Regulation of the rhizobactin 1021 regulon in Sinorhizobium meliloti in response to iron” 7th International Symposium on Microbial Iron Transport Storage and Metabolism, Institut Pasteur, Paris France.

IV Abbreviations, Units and Prefixes:

Abbreviations AmpR Ampicillin resistant CarbR Carbenicillin resistant CmR Chloramphenicol resistant GmR Gentamicin resistant KmR Kanamycin resistant SpeR Spectinomycin resistant TetR Tetracycline resistant Fr Ferrichrome Df Ferrioxamine B Rhz Rhizobactin 1021 Shz Schizokinen Cp Coprogen RtA Rhodoturilic acid Yb Yersiniabactin Hm Haemin Hb Haemoglobin MCS Multiple cloning site PCR Polymerase chain reaction SUST Single unit siderophore transporter SIT Siderophore iron transport ABC ATP-dependent binding cassette MFS Major facilitator superfamily TRAP Tripartite ATP-independent periplasmic ESR Extracytoplasmic solute receptor TCST Two-component signal transduction ECF Extracytoplasmic sigma factor nt Nucleotide LB Luria Bertani OD Optical density PAGE Polyacrylamide gel electrophoresis SDS Sodium dodecyl sulphate

V TEMED N, N, N, N’-Tetramethyl ethylenediamine IMAC Immobilised metal affinity chromatography FPLC Fast protein liquid chromatography pI Isoelectric point TMD Transmembrane Domain EPS exopolysaccharide

Units sec Second min Minute hr Hour rpm Revolutions per minute gav g-force (average) bp Base pair kb Kilo base psi Pounds per square inch Da Dalton M Molar mM Millimolar

Prefixes k Kilo (103) c Centi (10-2) m Milli (10-3) µ Micro (10-6) n Nano (10-9) p Pico (10-12)

VI Abstract

The bacterial cell membrane is the principal barrier for the acquisition of essential nutrients such as iron. Although critical to the survival of bacteria, iron is extremely limited in the environment due to the formation of insoluble hydroxides at physiological pH. To surmount these obstacles bacteria have evolved several mechanisms of iron acquisition. Sinorhizobium meliloti 2011, the endosymbiont of Medicago sativa, is capable of utilising several xenosiderophores and the haem compounds haemoglobin and haemin to overcome conditions of iron limitation. The high demand for iron in the nodule makes the study of S. meliloti 2011 biologically significant.

Two putative hydroxamate siderophore outer membrane receptors were identified on the S. meliloti 2011 chromosome. Insertional inactivation indicated that FhuA1 and FhuA2 function in the utilisation of ferrichrome and ferrioxamine B respectively. Analysis of the region directly downstream of FhuA2 resulted in the identification of a ferrioxamine B specific ferric iron reductase, fhuF (smc01658), and a periplasmic siderophore binding protein, fhuP (smc01659). FhuP was found to function with the inner membrane haem permease and ATPase, HmuU and HmuV respectively, to effect ferrichrome and ferrioxamine B utilisation. Insertions in hmuPSTUV resulted in a reduction in haem utilisation suggesting the presence of a secondary haem utlilisation system. Heterologous expression of HmuTUV but not HmuUV in an E. coli dppF mutant restored haem utilisation suggesting that all three proteins are required for haem utilisation and that HmuUV can function with two separate periplasmic binding proteins. Orthologues of HmuUV were cloned from Rhizobium leguminosarum bv. vicae 3841 and found to restore ferrichrome, ferrioxamine B and haem utlilisation in a S. meliloti hmuU mutant.

Previously it was demonstrated that Pseudomonas aeruginosa PAO1 is capable of utilising the xenosiderophores ferrichrome, ferrioxamine B and schizokinen. An inner membrane transporter, FoxB, was identified by heterologous expression in a S. meliloti siderophore transport deficient background. The expression and purification of full- length recombinant His-tagged FoxB was achieved.

VII Table of Contents

Chapter One Introduction 1.1: Introduction 2 1.2: Membrane Transport Superfamilies 4 1.2.1: The ATP-Binding Cassette (ABC) Superfamily 4 1.2.2: The Major Facilitator Superfamily (MFS) 5 1.2.3: The Tripartite ATP-Independent Periplasmic (TRAP) Transporter Family 6 1.3: Microbial Siderophores: Structure and Classification 9 1.3.1: Phenolates/Catecholates 9 1.3.2: Hydroxamates 10 1.3.3: Fungal Siderophores 13 1.4: Fe3+-Siderophore Active Transport in Gram Negative Bacteria 16 1.4.1: Outer Membrane Fe3+-Siderophore Receptors 17 1.4.2: Energy Coupled Active Transport: The TonB Complex 22 1.4.3: Delivery of Fe3+-Siderophores to the Cytoplasm by ABC-Transporters 26 1.4.4: Iron Release by Fe3+-Siderophore Reductase Activity 31 1.5: Bacterial Haem Acquisition 33 1.5.1: Haem Utilisation Systems: Inner and Outer Membrane Transport 33 1.5.2: Haemophores 36 1.5.3: Haem Oxygenase Facilitated Iron Release 37 1.5.4: Haem Transport via Dipeptide Transport Systems 38 1.6: Iron-Regulated Gene Expression 40 1.6.1: Fur 40 1.6.2: RirA 41 1.6.3: Mur 42 1.6.4: Irr 43 1.6.5: AraC-type Transcriptional Regulators 43 1.6.6: Two-Component Signal Transduction 44 1.7: Rhizobium-Legume Symbiosis 48 1.8: Iron Acquisition in The Rhizobia 51

VIII 1.8.1: Sinorhizobium meliloti 51 1.8.2: Rhizobium leguminosarum 52 1.8.3: Bradyrhizobium japonicum 53 1.9: Siderophore Mediated Iron Acquisition in Pseudomonas aeruginosa 55 1.9.1: Pyoverdine Utilisation 55 1.9.2: Pyochelin Utilisation 56 1.9.3: Xenosiderophore Utilisation 58 1.10: Iron Acquisition in Fungi 61 1.11: Summary 64

Chapter Two Materials & Methods 2.1: Bacterial Strains, Primer Sequences and Plasmids 66 2.2: Microbiological Media 74 2.3: Solutions and Buffers 78 2.4: Antibiotics 83 2.5: Isolation and Purification of DNA 83 2.5.1: Plasmid Preparation by the 1,2,3 Method 84 2.5.2: Preparation of Plasmid DNA by the Rapid Boiling Method 84 2.5.3: Plasmid DNA Isolation Using the GenElute Plasmid Miniprep Kit 85 2.5.4: Preparation of Total Genomic DNA from Sinorhizobium 85 2.5.5: Preparation of Total Genomic DNA Using the Wizard Genomic DNA Kit 86 2.5.6: Isolation of DNA from Agarose Gels 87 2.5.7: Purification and Concentration of DNA Samples 88 2.6: Quantitation of DNA and RNA 88 2.7: Agarose Gel Electrophoresis 88 2.8: In Silico Analysis of DNA and Protein Sequences 89 2.9: Competent Cells 90 2.9.1: Preparation of Competent Cells by RbCl Treatment 90 2.9.2: Preparation of Competent Cells by the TB Method 90 2.9.3: Preparation of Electrocompetent Cells 91 2.9.4: Transformation of Chemical Competent Cells 91 2.9.5: Electroporation of Electrocompetent Cells 92

IX 2.9.6: Determination of Competent Cell Efficiency 92 2.9.7: Sterilisation and Cleaning of Electroporation Cuvettes 92 2.10: Plant Nodulation and Nitrogen Fixation Assay 93 2.10.1: Surface Sterilisation of Medicago sativa 93 2.10.2: Nodulation Analysis of Medicago sativa 93 2.10.3: Acetylene Reduction Assay 94 2.10.4: Nodule Surface Sterilisation and Re-isolation of rhizobia 94 2.11: Bacterial Conjugation by Triparental Mating 95 2.12: Storing and Culturing of Bacteria 95 2.13: The Schaffner Weissmann Protein Assay 96 2.14: Membrane Protein Expression and Purification 97 2.14.1: Standard Expression Culture (Small Scale) 97 2.14.2: Membrane Isolation by Water Lysis 97 2.14.3: Large Scale Expression Culture 98 2.14.4: Cell Disruption 99 2.14.5: Isolation of Bacterial Membrane Fractions 99 2.14.6: Membrane Protein Purification 103 2.14.7: Standard Membrane Protein Solubilisation Procedure 103 2.14.8: Standard Membrane Protein IMAC Procedure 103 2.14.9: Recharging of Ni-NTA Resin 104 2.15: SDS-PAGE & Western Blotting of Membrane Proteins 104 2.15.1: Preparation of SDS Gels 104 2.15.2: Sample Preparation 105 2.15.3: Sample Application 105 2.15.4: Gel Staining 106 2.15.5: Gel Analysis 106 2.15.6: Western Blotting 107 2.16: Enzymatic Reactions 108 2.16.1: Enzymes and Buffers 108 2.16.2: RNase Reaction 108 2.16.3: Standard PCR Reaction Mixture 108 2.16.4: Standard PCR Program Cycle 108 2.16.5: Standard Phusion Taq PCR Program Cycle 108 2.16.6: 1 Kb DNA Molecular Marker 109

X 2.16.7: Klenow Reaction 109 2.16.8: T4 DNA Polymerase Reaction 110

Chapter Three Xenosiderophore Mediated Iron Acquisition by Sinorhizobium meliloti 2011 3.1: Introduction 112 3.2: Analysis of Xenosiderophore Utilisation by Sinorhizobium meliloti 2011 114 3.3: In Silico Analysis of the Sinorhizobium meliloti Genome in Search of Novel Siderophore Transport Systems 119 3.3.1: In Silico Analysis of FhuA1 120 3.3.2: In Silico Analysis of FhuA2 124 3.4: The Principles of Triparental Mating and the Mutagenesis of S. meliloti 129 3.5: Antibiotic Resistant Cassette Mutagenesis of fhuA1 and fhuA2 132 3.5.1: Mutagenesis of fhuA1 132 3.5.2: Mutagenesis of fhuA2 134 3.6: Xenosiderophore Utilisation by fhuA1 and fhuA2 Mutants 137 3.7: In Silico Analysis of the Genes Proximal to fhuA1 and fhuA2 139 3.7.1: In Silico Analysis of Smc01610 139 3.7.2: In Silico Analysis of Smc01658 141 3.7.3: In Silico Analysis of Smc01659 142 3.9.4: In Silico Analysis of Smc01660 144 3.8: Antibiotic Resistant Cassette Mutagenesis of smc01610 146 3.9: Xenosiderophore Utilisation by Mutant 2011rhtX-3smc01610::Tc 149 3.10: Complementation Analysis of the fhuA1 and smc01610 Mutants 150 3.10.1: Cloning of fhuA1 150 3.10.2: Cloning of smc01610 151 3.10.3: Cloning of smc01610 and fhuA1 152 3.10.4: Utilisation Phenotype of smc01610 and fhuA1 Mutants 153 3.11: Antibiotic Resistant Cassette Mutagenesis of smc01658, smc01659 and smc01660 155 3.11.2: Mutagenesis of smc01658 155 3.11.2: Mutagenesis of smc01659 157 3.11.3: Mutagenesis of smc01660 159

XI 3.12: Xenosiderophore Utilisation by smc01658, smc01659 and smc01660 Mutants 162 3.13: Complementation Analysis of the fhuA2, smc01658 and smc01659 Mutants 163 3.13.1: Cloning of fhuA2 163 3.13.2: Cloning of smc01658 164 3.13.3: Cloning of smc01659 164 3.13.4: Cloning of smc01658 and smc01659 165 3.13.5: Complementation Analysis of Xenosiderophore Utilisation Mutants 166 3.14: Analysis of Smc01658 Reductase Activity 169 3.14.1: Cloning of smc01658 and fhuF from E. coli 169 3.14.2: Substitution of E. coli FhuF with Smc01658 170 3.15: Analysis of the hmu Gene Cluster in S. meliloti 172 3.16: Transport of Ferrioxamine B and Ferrichrome by HmuUV 174 3.17: Expression of smc01659, hmuU and hmuV in a Heterologous Host 176 3.18: Effects of Xenosiderophore Utilisation Mutants on Symbiotic Nitrogen Fixation 178 3.19: Xenosiderophore Utilisation by R. leguminosarum and P. aeruginosa 181 3.19.1: Cloning hmuUV of R. leguminosarum bv vicae 3841 181

3.19.2: Expression of hmuUVRL in S. meliloti 2011rhtX-3hmuU::Tn5lacZ 182 3.19.3 Cloning phuUV of P. aeruginosa PAO1 182

3.19.4: Analysis of hmuUVRL and phuUV in a Heterologous Host 183 3.19.5: In silico Analysis of the R. leguminosarum bv vicae 3841 and P. aeruginosa PAO1 Genomes in Search of Smc01659 Homologues 184 3.20: Discussion 187

Chapter Four Haem Mediated Iron Acquisition by Sinorhizobium meliloti 2011 4.1: Introduction 194 4.2: Analysis of the hmuTUV Locus 195 4.2.1: In Silico Analysis of hmuTUV 195 4.2.2: Semi-Quantitative Analysis of Haem Uptake in hmuTUV mutants 199

XII 4.2.3: Haem Utilisation by hmuTUV Expressed in a Heterologous Host 200 4.3: In Silico Analysis of Dipeptide Transporters in S. meliloti 204 4.4: Antibiotic Resistant Cassette Mutagenesis of dppB1 and dppB2 207 4.4.1: Mutagenesis of dppB1 207 4.4.2: Mutagenesis of dppB2 209 4.4: Haem Utilisation by dppB1 and dppB2 Mutants 212 4.5: Expression of dppA1B1C1D1F1 and dppA2B2C2D2F2 in a Heterologous Host 213 4.5.1: Cloning of dppA1B1C1D1F1 and dppA2B2C2D2F2 213 4.5.2: Analysis of Haem Utilisation by dppA1B1C1D1F1 and dppA2B2C2D2F2 Expressed in a Heterologous Host 214 4.6: Discussion 216

Chapter Five FoxB of Pseudomonas aeruginosa PAO1: Protein Expression and Purification 5.1: Introduction 221 5.2: In Silico Topology Analysis of Membrane Proteins 223 5.2.1: In Silico Analysis of FoxB Topology 223 5.2.2: In Silico Analysis of RhtX Topology 224 5.2.3: In Silico Analysis of HmuU Topology 225 5.3: Cloning of Affinity Tagged Recombinant Membrane Proteins 227 5.4: Small Scale Expression and Purification of Membrane Proteins 230 5.5: Large Scale Expression and Purification of Histidine-Tagged FoxB 235 5.6: Solubilisation and Purification of Histidine-Tagged FoxB 238 5.6.1: Solubilisation of FoxB 238 5.6.2: Standard IMAC Purification 238 5.6.3: Fast Protein Liquid Chromatography (FPLC) of FoxB 240 5.6.4: Optimised IMAC of FoxB 244 5.7: Discussion 246

Concluding Remarks 6.1: Concluding Remarks 252

XIII References 255

XIV List of Figures

Figure 1.1: Secondary Active Transport Systems in Bacteria 6 Figure 1.2: Model for ABC, TRAP and MFS Transport Systems 8 Figure 1.3: Structure of Enterobactin 9 Figure 1.4: Structure of Aerobactin 11 Figure 1.5: Structure of Rhizobactin 1021 11 Figure 1.6: Structure of Desferrioxamine B and Desferrioxamine E 12 Figure 1.7: Structure of Rhodotorulic acid 13 Figure 1.8: Structure of Dimerumic acid 13 Figure 1.9: Structure of Coprogen 14 Figure 1.10: Ferrichrome Siderophores 15 Figure 1.11: Genetic Arrangement of Genes Encoding fhuACDB of E. coli and Orthologues 17 Figure 1.12: Crystal Structures of FhuA, FepA and FecA 21 Figure 1.13: Overall Structure of the TonB-FhuA Complex 24 Figure 1.14: Crystal Structure of FhuD 28 Figure 1.15: A Model for Siderophore Utilisation 30 Figure 1.16: Structure of Haem 33 Figure 1.17: Mechanism of Haem Uptake in Gram Negative Bacteria 34 Figure 1.18: Structure of the Serratia marcescens HasA Haemophore 37 Figure 1.19: Overall Structure of Haem Oxygenase (A) HemO from N. meningitides and (B) IsdG from S. aureus 38 Figure 1.20: Crystal Structure of P. aeruginosa Fur 41 Figure 1.21: The Ferric Citrate Transport and Regulatory Systems 46 Figure 1.22: Early Signal Exchange between Alfalfa (M. sativa) and S. meliloti 49 Figure 1.23: The Infection Thread Leading to the Development of the Symbiosome 50 Figure 1.24: The FpvA-Pvd-Fe Crystal Structure 56 Figure 1.25: Two-Dimensional Topology of FptA 57 Figure 1.26: Specificity of Transporters of Siderophore-Iron Transporters in S. cerevisiae 62 Figure 2.1: pCR2.1 Vector 71 Figure 2.2: pBBR1MCS-5 Vector 72

XV Figure 2.3: pUC4K vector 72 Figure 2.4: pJQ200sk+ vector 73 Figure 2.5: pTTQ18 vector 73 Figure 2.5: Isolation of Bacterial Membrane Fractions Methodology Flow Chart 100 Figure 2.6: Sucrose Gradient 102 Figure 2.7: Assembly of SDS Gel, Membrane and Filter Paper for Western Blotting 107 Figure 3.1: Organisation and Mutagenesis of the Rhizobactin 1021 Regulon 113 Figure 3.2: Utilisation of Xenosiderophores by 2011rhbA62 116 Figure 3.3: Analysis of Coprogen Utilisation by S. meliloti 2011rhtX3 and Rm818 117 Figure 3.4: Multiple Sequence Alignment of E. coli K12 FhuA 120 Figure 3.5: Amino Acid Sequence of FhuA1 121 Figure 3.6: Multiple Sequence Alignment of S. meliloti 2011 (FhuA1) 122 Figure 3.7: TonB Region I Motif 123 Figure 3.8: TonB Region II Motif 123 Figure 3.9: TonB Region III Motif 123 Figure 3.10: Genetic Organisation of the fhuA1 Region of S. meliloti 124 Figure 3.11: Amino Acid Sequence of FhuA2 125 Figure 3.12: Multiple Sequence Alignment of S. meliloti 2011 (FhuA2) 126 Figure 3.13: TonB Region I Motif 127 Figure 3.14: TonB Region II Motif 127 Figure 3.15: TonB Region III Motif 127 Figure 3.16: Genetic Organisation of the fhuA2 Region of S. meliloti 128 Figure 3.17: Schematic of Triparental Mating 130 Figure 3.18: Schematic of Homologous Recombination 131 Figure 3.19: PCR Primers for the Amplification of a Region Encoding fhuA1 132 Figure 3.20: Analysis of the fhuA1 Mutant by PCR 134 Figure 3.21: PCR Primers for the Amplification of a Region Encoding fhuA2 134 Figure 3.22: Analysis of the fhuA2 Mutant by PCR 136 Figure 3.23: Analysis of Siderophore Utilisation in 2011rhtX-3fhuA1::Km and 2011rhtX-3fhuA2::Km 137 Figure 3.24: Amino Acid Sequence of Smc01610 139

XVI Figure 3.25: Multiple Sequence Alignment of S. meliloti 2011 (Smc01610) 140 Figure 3.26: Prompter Region of Smc01610 141 Figure 3.27: Amino Acid Sequence of Smc01658 141 Figure 3.28: Multiple Sequence Alignment of S. meliloti 2011 (Smc01658) 142 Figure 3.29: Multiple Sequence Alignment of the [2Fe-2S]Cys4 Motif of S. meliloti 2011 (Smc01658) 142 Figure 3.30: Amino Acid Sequence of Smc01659 143 Figure 3.31: Multiple Sequence Alignment of S. meliloti 2011 (Smc01659) 144 Figure 3.32: Amino Acid Sequence of Smc01660 144 Figure 3.33: Multiple Sequence Alignment of S. meliloti 2011 (Smc01660) 145 Figure 3.34: PCR Primers for the Amplification of a Region Encoding smc01610 146 Figure 3.35: Confirmation of Mutant 2011rhtX-3smc01610::Tc 148 Figure 3.36: PCR Primers for the Amplification of fhuA1 for Cloning and Expression 151 Figure 3.37: PCR Primers for the Amplification of smc01610 for Cloning and Expression 151 Figure 3.38: PCR Primers for the Amplification of smc01610-fhuA1 for Cloning and Expression 152 Figure 3.39: Mutagenesis and Complementation of smc01610 and fhuA1 153 Figure 3.40: PCR Primers for the Amplification of a Region Encoding smc01658 155 Figure 3.41: Analysis of the smc01658 Mutant by PCR 157 Figure 3.42: PCR Primers for the Amplification of a Region Encoding smc01659 157 Figure 3.43: Analysis of the smc01659 Mutant by PCR 159 Figure 3.44: PCR Primers for the Amplification of a Region Encoding smc01660 159 Figure 3.45: Analysis of the smc01660 Mutant by PCR 161 Figure 3.46: PCR Primers for the Amplification of fhuA2 for Cloning and Expression 163 Figure 3.47: PCR Primers for the Amplification of smc01658 for Cloning and Expression 164

XVII Figure 3.48: PCR Primers for the Amplification of smc01659 for Cloning and Expression 165 Figure 3.49: PCR Primers for the Amplification of smc01658-smc01659 for Cloning and Expression 166 Figure 3.50: Mutagenesis and Complementation of fhuA2 Region 167 Figure 3.51: PCR Primers for the Amplification of fhuF of E. coli 170 Figure 3.52: Organisation and Mutagenesis of the hmu Gene Cluster 173 Figure 3.53: Nodulation Analysis of M. sativa Seedlings by S. meliloti 2011 179 Figure 3.54: Nodule Formation Induced by S. meliloti 2011 on a 30 Day Old M. sativa Seedling 179 Figure 3.55: PCR Primers for the Amplification of hmuUV of R. leguminosarum bv vicae 3841 181 Figure 3.56: PCR Primers for the Amplification of phuUV of P. aeruginosa PAO1 183 Figure 3.57: Multiple Sequence Alignment of S. meliloti 2011 Smc01659 and R. leguminosarum bv vicae 3841 RL2713 185 Figure 3.58: Multiple Sequence Alignment of S. meliloti 2011 (Smc01659) 186 Figure 3.59: A Model of Hydroxamate Siderophore Utilisation and Assimilation in S. meliloti 2011 191 Figure 4.1: Global Multiple Sequence Alignment of S. meliloti 2011 HmuU 197 Figure 4.2: Utilisation of Haemin by S. meliloti 2011rhtX3 and hmuTUV Mutants 200 Figure 4.3: Analysis of Haemin and Haemoglobin Utilisation by E. coli FB827dppF::Km Expressing S. meliloti 2011 hmuTUV Genes with S. marcesence hasR 202 Figure 4.4: Multiple Sequence Alignment of E. coli K12 DppB 205 Figure 4.5: PCR Primers for the Amplification of a Region Encoding dppB1 207 Figure 4.6: Analysis of the dppB1 Mutant by PCR 209 Figure 4.7: PCR Primers for the Amplification of a Region Encoding dppB2 210 Figure 4.8: Analysis of the dppB2 Mutant by PCR 211 Figure 4.9: PCR Primers for the Amplification of dppA1B1C1D1F1 and dppA2B2C2D2F2 for Cloning 214 Figure 5.1: Prediction of Transmembrane Helices in FoxB 224 Figure 5.2: Prediction of Transmembrane Helices in RhtX 225

XVIII Figure 5.3: Prediction of Transmembrane Helices in HmuU 226 Figure 5.4: PCR Primers for the Amplification of foxB, rhtX and hmuU for Expression 228 Figure 5.5: Amino Acid Sequence of Recombinant FoxB 228 Figure 5.6: Amino Acid Sequence of Recombinant RhtX 229 Figure 5.7: Amino Acid Sequence of Recombinant HmuU 229 Figure 5.8: Expression of FoxB, RhtX and HmuU in E. coli BL21(DE3) 230 Figure 5.9: SDS-PAGE Gel of Mixed Membrane Preparation of Expressed Histidine-Tagged RhtX, FoxB and HmuU Membrane Proteins 232 Figure 5.10: Standard Curve for the Determination of Migrated Distance of Membrane Proteins on SDS Gels 232 Figure 5.11: Western Blot Analysis of Mixed Membranes Prepared from E. coli BL21(DE3) 234 Figure 5.12: Standard Curve for the Determination of Migrated Distance of Membrane Proteins on Western blot 234 Figure 5.13: SDS-PAGE Gel of Isolated Membrane Fractions of an E. coli Strain Expressing Histidine-Tagged FoxB 236 Figure 5.14: FoxB Purification - Western Blot Analysis of Isolated Membranes Fractions 237 Figure 5.15: IMAC Purification of Histidine-Tagged FoxB 239 Figure 5.16: FoxB Purification - Western Blot Analysis of Standard IMAC Membranes Fractions 240 Figure 5.17: FPLC Stepped Elution of FoxB 242 Figure 5.18: Coomassie Stained SDS Gel of FPLC Fractions 243 Figure 5.19: Silver Stained SDS Gel of FPLC Fractions 243 Figure 5.20: Coomassie Stained Gel and Western Blot of Purified FoxB 245

XIX List of Tables

Table 2.1: Bacterial Strains 66 Table 2.2: Primer Sequences 68 Table 2.3: Plasmids 69 Table 2.4: Schaffner Weissmann Protein Standards Range 96 Table 2.5: Preparation of SDS Gels 105 Table 2.6: SDS-PAGE Molecular Weight Markers 106 Table 3.1: Optimal Siderophore Concentrations for S. meliloti 2011 115 Table 3.2: Siderophore Utilisation Analysis in S. meliloti 116 Table 3.3: S. meliloti Proteins Displaying Significant Homology to FhuA of E. coli K12 119 Table 3.4: Proteins Displaying Significant Homology to FhuA1 121 Table 3.5: Proteins Displaying Significant Homology to FhuA2 125 Table 3.6: PCR Conditions for the Amplification of the Region Encoding fhuA1 133 Table 3.7: PCR Conditions for the Amplification of the Region Encoding fhuA2 135 Table 3.8: Analysis of Siderophore Utilisation in 2011rhtX-3fhuA1::Km and 2011rhtX-3fhuA2::Km 137 Table 3.9: Proteins Displaying Significant Homology to Smc01610 140 Table 3.10: Proteins Displaying Significant Homology to Smc01658 141 Table 3.11: Proteins Displaying Significant Homology to Smc01659 143 Table 3.12: Proteins Displaying Significant Homology to Smc01660 145 Table 3.13: PCR Conditions for the Amplification of the Region Encoding smc01610 146 Table 3.14: Analysis of Siderophore Utilisation in 2011rhtX-3smc01610::Tc 149 Table 3.15: PCR Conditions for the Amplification of fhuA1 for Cloning 151 Table 3.16: PCR Conditions for the Amplification of smc01610 for Cloning 152 Table 3.17: PCR Conditions for the Amplification of smc01610-fhuA1 for Cloning 153 Table 3.18: Utilisation of Ferrioxamine B and Ferrichrome by Mutant Transconjugants 154

XX Table 3.19: PCR Conditions for the Amplification of the Region Carrying smc01658 156 Table 3.20: PCR Conditions for the Amplification of the Region Encoding smc01659 158 Table 3.21: PCR Conditions for the Amplification of the Region Carrying smc01660 160 Table 3.22: Analysis of Siderophore Utilisation in 2011rhtX-3smc01658::Km, 2011rhtX-3smc01659::Km and 2011rhtX-3smc01660::Km 162 Table 3.23: PCR Conditions for the Amplification of fhuA2 for Cloning 163 Table 3.24: PCR Conditions for the Amplification of smc01658 for Cloning 164 Table 3.25: PCR Conditions for the Amplification of smc01659 for Cloning 165 Table 3.26: PCR Conditions for the Amplification of smc01658-smc01659 for Cloning 166 Table 3.27: Iron Utilisation in Transconjugants 167 Table 3.28: PCR Conditions for the Amplification of fhuF of E. coli 170 Table 3.29: Analysis of Smc01658 Reductase Activity in a Heterologous Host 171 Table 3.30: Analysis of Siderophore Utilisation by 2011rhtX-3hmuT::Km 174 Table 3.31: Analysis of Siderophore Utilisation by 2011rhtX-3hmuU::Tn5lac and 2011rhtX-3hmuV::Km 175 Table 3.32: Siderophore Utilisation by E. coli B1713 177 Table 3.33: Effect of Mutants on Symbiotic Nitrogen Fixation 178 Table 3.34: PCR Conditions for the Amplification of hmuUV of R. leguminosarum bv vicae 3841 182 Table 3.35: Analysis of Siderophore Utilisation by 2011rhtX-3hmuU::Tn5lac

expression hmuUVRL 182 Table 3.36: PCR Conditions for the Amplification of phuUV of P. aeruginosa PAO1 183

Table 3.37: Analysis of hmuUVRL and phuUV in E. coli B1713 184 Table 3.38: Pseudomonadaceae Proteins Displaying Significant Homology to Smc01659 186 Table 4.1: Proteins Displaying Significant Homology to HmuU 196 Table 4.2: In Silico Analysis of the hmuTUV Locus of S. meliloti 198 Table 4.3: Haemin and Haemoglobin Utilisation by E. coli FB827dppF::Km 202

XXI Table 4.4: S. meliloti Proteins Displaying Significant Homology to DppB of E. coli K12 204 Table 4.5: In Silico Analysis of the DppA1B1C1D1F1 Locus of S. meliloti 2011 206 Table 4.6: PCR Conditions for the Amplification of the Region Encoding dppB1 208 Table 4.7: PCR Conditions for the Amplification of the Region Encoding dppB2 210 Table 4.8: Analysis of Haemin and Haemoglobin Utilisation in 2011rhtX-3hmuTdppB1::Tc and 2011rhtX-3hmuTdppB2::Tc 212 Table 4.9: PCR Conditions for the Amplification of dppA1B1C1D1F1 and dppA2B2C2D2F2 for Cloning 214 Table 4.10: Haemin and Haemoglobin Utilisation by E. coli FB827dppF::Km 215 Table 5.1: Mixed Membrane Protein Concentrations 231 Table 5.2: Calculated Migrated Distance of Induced Protein Bands on SDS-PAGE Gel 233 Table 5.3: Calculated Migrated Distance of RhtX and FoxB on Western Blot 234 Table 5.4: FoxB Purification - Isolated Membrane Protein Concentrations 236 Table 5.5: Wash and Elution Buffer for FPLC 241