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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 fulllength 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
1.3:
6
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
1.4.4:
1.5:
ABC-Transporters
26
Iron Release by Fe3+-Siderophore Reductase Activity
31
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
2.5.5:
Preparation of Total Genomic DNA Using the Wizard Genomic
85
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
4.2.2:
Semi-Quantitative Analysis of Haem Uptake in hmuTUV mutants 199
XII
195
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
4.5:
Expression of dppA1B1C1D1F1 and dppA2B2C2D2F2 in a Heterologous
Host
213
4.5.1:
Cloning of dppA1B1C1D1F1 and dppA2B2C2D2F2
4.5.2:
Analysis of Haem Utilisation by dppA1B1C1D1F1
and dppA2B2C2D2F2 Expressed in a Heterologous Host
4.6:
212
Discussion
213
214
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
References
255
XIII
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
XIV
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
Figure 3.24:
2011rhtX-3fhuA2::Km
137
Amino Acid Sequence of Smc01610
139
XV
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
Figure 3.37:
151
PCR Primers for the Amplification of smc01610 for Cloning and
Expression
Figure 3.38:
151
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
Figure 3.47:
163
PCR Primers for the Amplification of smc01658 for Cloning and
Expression
164
XVI
Figure 3.48:
PCR Primers for the Amplification of smc01659 for Cloning and
Expression
Figure 3.49:
165
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
Figure 3.55:
179
PCR Primers for the Amplification of hmuUV of R. leguminosarum
bv vicae 3841
Figure 3.56:
181
PCR Primers for the Amplification of phuUV of
P. aeruginosa PAO1
Figure 3.57:
183
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
Figure 4.3:
200
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
XVII
Figure 5.3:
Prediction of Transmembrane Helices in HmuU
Figure 5.4:
PCR Primers for the Amplification of foxB, rhtX and hmuU for
226
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
Figure 5.10:
Standard Curve for the Determination of Migrated Distance of
Membrane Proteins on SDS Gels
Figure 5.11:
234
Standard Curve for the Determination of Migrated Distance of
Membrane Proteins on Western blot
Figure 5.13:
234
SDS-PAGE Gel of Isolated Membrane Fractions of an E. coli
Strain Expressing Histidine-Tagged FoxB
Figure 5.14:
232
Western Blot Analysis of Mixed Membranes Prepared from E. coli
BL21(DE3)
Figure 5.12:
232
236
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
XVIII
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
Table 3.7:
133
PCR Conditions for the Amplification of the Region Encoding
fhuA2
Table 3.8:
135
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
Table 3.18:
153
Utilisation of Ferrioxamine B and Ferrichrome by Mutant
Transconjugants
154
XIX
Table 3.19:
PCR Conditions for the Amplification of the Region Carrying
smc01658
Table 3.20:
156
PCR Conditions for the Amplification of the Region Encoding
smc01659
Table 3.21:
158
PCR Conditions for the Amplification of the Region Carrying
smc01660
Table 3.22:
160
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
Table 3.31:
Analysis of Siderophore Utilisation by 2011rhtX-3hmuU::Tn5lac
174
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
Table 3.35:
Analysis of Siderophore Utilisation by 2011rhtX-3hmuU::Tn5lac
expression hmuUVRL
Table 3.36:
182
182
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
XX
Table 4.4:
S. meliloti Proteins Displaying Significant Homology to DppB of
E. coli K12
Table 4.5:
204
In Silico Analysis of the DppA1B1C1D1F1 Locus of
S. meliloti 2011
Table 4.6:
206
PCR Conditions for the Amplification of the Region Encoding
dppB1
Table 4.7:
208
PCR Conditions for the Amplification of the Region Encoding
dppB2
Table 4.8:
210
Analysis of Haemin and Haemoglobin Utilisation in
2011rhtX-3hmuTdppB1::Tc and 2011rhtX-3hmuTdppB2::Tc
Table 4.9:
212
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
Table 5.2:
Calculated Migrated Distance of Induced Protein Bands on
SDS-PAGE Gel
231
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
XXI