....................................................................................................................................................................... ii Preface.......................................................................................................................................................................... iv Table
of
Contents.........................................................................................................................vii List
of
Tables ............................................................................................................................................................. xi List
of
Figures...........................................................................................................................................................xii List
of
Symbols,
Abbreviations
and
Nomenclature.................................................................................xiv Acknowledgements ............................................................................................................................................ xvii Chapter
1.
Introduction ......................................................................................................................................... 1 1.1.
Staphylococcus
aureus ................................................................................................................................ 1 1.2.
Iron
in
the
human
host .............................................................................................................................. 2 1.3.
Iron
uptake
in
Gram-‐negative
bacteria .............................................................................................. 4 1.4.
Iron
uptake
in
Gram-‐positive
bacteria ............................................................................................ 10 1.5.
S.
aureus
iron
uptake
systems ............................................................................................................. 11 1.5.1.
The
S.
aureus
heme
uptake
system............................................................................................ 12 1.5.2.
Siderophore
uptake
systems........................................................................................................ 16 1.6.
Objectives
of
this
thesis.......................................................................................................................... 20 Chapter
2.
Methods............................................................................................................................................... 22 2.1.
S.
aureus
growth
conditions ................................................................................................................. 22 2.2.
Construction
of
S.
aureus
mutant
strains........................................................................................ 22 2.2.1.
Construction
of
S.
aureus
isdA::Km............................................................................................ 22 2.2.2.
Construction
of
S.
aureus
isdE::Km............................................................................................ 23 2.2.3.
Site-‐directed
mutagenesis
of
S.
aureus
isdE
for
in
vivo
assays....................................... 23 2.3.
Heme
plate
bioassays.............................................................................................................................. 24 2.4.
Heme-‐dependent
bacterial
growth
studies
in
liquid
culture................................................. 24 2.5.
Peroxidase
staining.................................................................................................................................. 24 2.6.
Recombinant
protein
expression
systems
for
non-‐crystallographic
use......................... 25 2.6.1.
GST-‐IsdA
site
directed
mutagenesis
and
expression
for
heme
scavenging
from
E. coli........................................................................................................................................................................ 25 2.6.2.
Cloning
and
IsdE
protein
expression
for
heme
scavenging
from
E.
coli ................... 26 2.6.3.
Purification
of
staphyloferrin
A
biosynthetic
enzymes.................................................... 27 2.7.
UV/Visible
absorption
spectroscopic
characterization
of
heme
binding
from
the cytoplasm
of
E.
coli ........................................................................................................................................... 27 2.8.
Staphyloferrin
A
purification
from
culture
supernatants ....................................................... 28 2.9.
Staphyloferrin
A
in
vitro
synthesis.................................................................................................... 29 2.10.
Determination
of
ferric-‐staphyloferrin
A
concentration ...................................................... 29 2.11.
Fluorescence
spectroscopy
siderophore
titrations................................................................. 30 2.12.
Cloning,
expression
and
purification
for
structure
determination .................................. 30 2.12.1.
Expression
of
IsdAN
and
IsdAN
variants ............................................................................... 31 2.12.2.
Protein
expression
of
IsdE
for
structure
determination............................................... 32 2.12.3.
Cloning
and
protein
expression
of
HtsA
for
structure
determination .................... 32 2.13.
Crystallization
and
structure
determination ............................................................................. 33