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Diss. ETH
hemoglobin (VHb)
From Vitreoscilla
class of
14619
no
protective
and
to
a
growth promoting
hemoglobin proteins
bacterial
A dissertation submitted to the
SWISS FEDERAL INSTITUTE OF TECHNOLOGY
for the
degree
of
Doctor of Natural Sciences
Presented
by
Alexander Daniel
Dipl.
Frey
Natw. ETH, ETH Zürich
Born March
3rd, 1974
Citizen of Auenstein, AG
Accepted
on
the recommendation of
Prof. Dr. B. Witholt, examiner
Prof. Dr. H.
Häggman,
PD Dr. P. T.
co-examiner
Kallio, co-examiner
2002
novel
ZÜRICH
Table of contents
Table of contents
Summary
Summary
1
Zusammenfassung
3
5
Chapter
1
General introduction and literature overview
Chapter
2
Expression
Chapter
3
Dissection
Alcaligenes eutrophus flavohemoprotein and
engineered Vitreoscilla hemoglobin-reductase fusion
protein for improved hypoxic growth of Escherichia coli
50
of
75
of
of
the
central
carbon
metabolism
13C NMR flux
hemoglobin-expressing Escherichia coli by
distribution analysis in microaerobic bioprocesses
Chapter 4
Bacterial
hemoglobins
and
flavohemoglobins
for
97
alleviation of nitrosative stress in Escherichia coli
Expression of Vitreoscilla hemoglobin in hybrid aspen
(Populus tremula x tremuloides): an attempt to improve
growth of an economically relevant tree species
120
Conclusions
Conclusions and final remarks
149
Biography
Curriculum vitae
151
Chapter
5
I
Summary
Summary
hemoglobin (VHb)
Vitreoscilla
biotechnological
relevant
of these
productivity
argued
growth
conditions
as
encountered in
Flavohemoglobin protein
N-terminal
tail
protein
hemoglobin
can
and
non-linked
a
ability
and
lacking
an
and FHP
Interestingly,
typical
physiological
was
an
In
a
FAD-
contrast
to
intramolecular reductase domain. However,
reductase has been identified
highly homologous
protein
to
the
hemoglobin
fusions
protein
flavohemoglobin
VHb-Red
relative to the native VHb
production
of
or
domain
of VHb and the
structures of FHP
were
engineered.
of FHP resulted in
protein in microaerobic
a
recombinant reporter
using
cells
fractional
expressing
cells had
basis of the observed effects
of the metabolism of E. coli cells
for anaerobiosis,
expressing
consists of
increased.
in-depth analysis
FHPg
ideally
proteins
modules:
domain.
(NAD)
binding
several chimeric
of either the artificial
To understand the
an
not
are
The redox-active
functionally separate protein
Escherichia coli cells. Furthermore,
was
protein
promote growth under oxygen limited
NADH-dependent methemoglobin
significant growth improvements
protein
of this
and
eukaryotic origin.
eutropha (Alcaligenes eutrophus)
reductase domain of FHP and truncated
growing
properties
and
C-terminal reductase domain.
a
NAD(P)H
(FHPg). Therefore,
Expression
prokaryotic
growth
large-scale production bioprocesses.
in its native host Vitreoscilla. VHb is
of FHP
to
of Ralstonia
VHb is
flavohemoglobins,
such
that the
be divided into two
(FAD)
binding
hosts from
various
in
limitation from all host cells. Therefore, novel
screened to test their
were
successfully expressed
been
which resulted in the enhanced
organisms,
heterologous
However, it has been
suited to relieve
has
a
13C-labeling
VHb
showing
a
or
and
a
functional TCA
lower in these cells indicative of
have
performed
expressing VHb, VHb-Red,
2-dimensional NMR
VHb-Red revealed
branched TCA
we
cycle.
a
metabolic
technique.
configuration
In contrast, FHP
or
FHPg
cycle. Furthermore, glucose consumption
more
1
efficient carbon utilization. All studied
Summary
strains showed
a
higher
ATP content and
an
that
flavohemoglobins
are
increase
ATP/ADP
ratio relative to
control cells.
Due to the
findings
NO' and their
have
expression results
the
analyzed
possibility
if
also able to mediate this stress
VHb,
also
were
able
in the
relieving
of cells from nitrosative stress,
and
flavohemoglobins
cell
under
growth
hemoglobins
limited.
is
the NO*
importance of
this
By engineering
degrading activity
protein
a
was
trees
was
VHb-expression
on a
followed under various
few
on
the
characteristics
that VHb
are
stress
of
significantly enhanced, showing
the
the
to
trees have been
growth conditions,
of
plant
differences
cells contain
a
indeed able to either
more
as
of
in
such
generated
higher
material
as
amount of
2
to
analyze
Growth of these
different temperature
well
and
growth
photosynthesize
efficiently.
as
VHb-expression
observed. However, ultrastructural
expressing
these lines
the carbon
were
nitrosative
complex, perennial plant species.
genetic background
significant
statistically
such
C-terminus
reductase
and UV-B illumination. However, the effect of
dependent
are
domain in NO' detoxification.
Furthermore, VHb-expressing hybrid aspen
the effect of
we
degrading capacity
conditions. However, in vitro assays revealed that the NO'
hemoglobins
general
in
Interestingly, hemoglobins,
effect.
normal
retain
to
protection
hemoglobins
in the detoxification of
key players
as on
other
analysis
growth
on
the line.
was
Only
phenotypical
of cells revealed
starch, demonstrating that
more
efficiently
or
to utilize
Summary
Zusammenfassung
Vitreoscilla
Organismen
Die
exprimiert.
Wachstumscharakteristika
Zellen.
eukaryotischen
Eigenschaften
von
VHb
Fähigkeit,
Produktivität
nicht
jedoch
allen
in
verbesserten
in
und
prokaryotischen
in
bekannt
biochemischen
die
ob
heterologen Organismen geeignet sind,
aufzuheben. Daher wurden
Wachstumslimitierungen
ihre
ist
resultierte
Expression
erhöhter
und
Es
biotechnologisch wichtigen
wurde in verschiedenen
Hämoglobin (VHb)
Wachstum unter Sauerstoff limitierten
um
Proteine evaluiert
neue
Bedingungen
zu
um
fördern,
zu
testen.
Das
Flavohämoglobinprotein
aus
einem
N-terminalen
aus
Ralstonia
Hämoglobin
Reduktasedomäne besteht
aus
bindendes
NAD(P)H
den
(FAD)
und ein
Flavohämoglobinen,
Proteindomäne. Eine
eutropha (Alcaligenes eutrophus)
und
einer
zwei funktionell
fehlt
VHb
C-terminalen Reduktase.
unabhängigen
bindendes
(NAD)
dazugehörende Methämoglobin
Modul. Im
Gegensatz
FHP
von
zu
redox-aktive
Reduktase wurde
Hämoglobindomäne
Vitreoscilla identifiziert. VHb and die
Die
Modulen: ein FAD
intramolekulare
eine
besteht
jedoch
(FHPg)
in
sind
strukturell stark verwandt. Daher wurden verschiedene Proteinfusionen zwischen
VHb und den
von
im
FHP
Komponenten
hergestellt.
Vergleich
zu
der Reduktase
Das künstliche
von
FHP als auch verkleinerte Formen
Flavohämoglobin
VHb das Wachstum
von
VHb-Red und FHP konnten
Escherichia coli
Zusätzlich wurde die Produktion eines rekombinanten
am
stärksten stimulieren.
Reporterproteins
in diesen
Zellen stark erhöht.
Um die beobachteten Effekte besser verstehen
von
E. coli
mittels
können, wurde der Stoffwechsel
Zellen, die entweder VHb, VHb-Red, FHP oder FHPg exprimierten,
13C-Markierung
analysiert.
zu
In
und
einer
2-dimensionalen
NMR
Methode
genauer
Zellen, die VHb oder VHb-Red exprimieren, konnten festgestellt
werden, dass der Zitronensäurezyklus unterbrochen ist, d.h. eine für anaerobe
Bedingungen typische
Zitronensäurezyklus
Konstellation
aktiv in FHP oder
auftritt.
Im
Gegensatz
FHPg exprimierenden
3
dazu
war
der
Zellen. Zusätzlich
Summary
war
die Glukoseaufnahme in diesen Zellen tiefer,
Stoffwechsel hindeutet. Alle rekombinante
wiesen im
Vergleich
zu
ATP/ADP Verhältnis
Flavohämoglobine
schützt Zellen
auf einen effizienteren
Hämoproteine exprimierenden
Zellen
Kontrollen einen höheren ATP Gehalt und ein besseres
auf.
sind sehr
vor
was
wichtig
für den Abbau
der schädlichen
untersucht, ob Flavohämoglobine
NO* und ihre
generellen
und auch
Expression
Wir haben
nitrosativen Stresses.
Wirkung
im
von
Hämoglobine
diese
Hämoglobine
konnten ein
normales Zell Wachstum unter nitrosativem Stress erhalten. In vitro
Experimente
schützende
hingegen
Eigenschaften
zeigten,
besitzen. VHb und andere
dass
Fähigkeit
die
Anfügen
eingeschränkt
ist. Durch das
Hämoglobins
kann der NO'-Abbau
Wichtigkeit dieser
VHb
komplexen, mehrjährigen
wurde
unter
Temperaturen,
Entgiftung von
zu
generiert,
und in anderen
Nur
war
der Zellstruktur
höheren
Stärkegehalt
dass die transgenen
den C-Terminus eines
was
die
NO' demonstriert.
den Effekt
VHb in einer
von
Das Wachstum dieser Bäume
verschiedenen
Strahlung, verfolgt.
Der Effekt der
vom
d.h.
als auch
genetischen Hintergrund
wenige signifikante
zeigte hingegen,
Hämoglobinen
bei
phänotypischen Eigenschaften
Analyse
bei
erhöht werden,
Wachstumsbedingungen,
auf das Wachstum
abhängig.
um
analysieren.
normaler und erhöhter UV-B
der Zellline
an
jedoch signifikant
wurden
Pflanze
verschiedenen
VHb-Expression
von
einer Reduktase
Proteindomäne für die
exprimierende Pappeln
abzubauen
NO'
Unterschiede im Wachstum
festgestellt
werden. Die
exprimierende
Zellen einen
konnten
dass VHb
besitzen als nichttransformierte Zellen. Dies ist ein Hinweis,
Pappeln
in der
oder den Kohlenstoff effizienter
zu
Lage sind,
benutzen.
4
Kohlenstoff effizienter
zu
fixieren
Chapter
1: Introduction
General introduction and literature overview
1: Introduction
Chapter
of oxygen for life
Significance
The
of oxygen relates to both its
importance
secondary
effects
metabolism and
on
cellular metabolism to work at its
to
regulation
of
availability
obligate
can
affect the
optimal
level
absence of oxygen
or
or
use
regarding
which
are
as a
substrate and its
a
substrate offers the
substrate utilization and
expressed
the presence of toxic
or
as
lifestyle. Oxygen
or
in response to the
which
by-products,
free radicals
produced
of
sets of
specific
proteins. Second, they
can
affect
are
from it
they
affect bacterial cells in at least two ways. First,
indirectly
biosynthesis
Its
use
The latter effect of oxygen includes the
variety of cellular functions,
a
consequences of aerobic
directly
physiology.
optimal yield.
energy at its
gain
primary
can
protein
function.
coli,
In Escherichia
two
groups of
in these two different set of
anaerobic conditions
and
OxyR
are
or
vice
global regulatory proteins
physiological
involved in the
generally
involved
conditions: switches from aerobic to
regulated by
versa are
are
global regulation
FNR and ArcAB, whereas SoxRS
of genes,
expressed
in the response
to oxidative stress conditions.
intensely
The molecule most
hemoglobin. Hemoglobin
binding protein,
oxygen-binding
But
recent
hemoglobins
most
molecule has for
prove
plants,
and its
Oxygen sensing
Oxygen
is the
by
mankind is
studied and best characterized
oxygen-
both at functional and molecular level. The presence of this
findings
nonvertebrates,
associated with utilization of oxygen
and
the
long
been
all
most
bacteria.
implications
on
presence of oxygen. The
The
ubiquitous
of
state
adaptation
acceptor of choice when
use
to
be restricted to mammals.
existence
knowledge
in
mammals,
on
bacterial
cellular metabolism is discussed below.
and molecular
is the electron
thought
to
hypoxia
E. coli cells
in E. coli
are
grown in the
of oxygen allows the cellular metabolism to
6
generate
Chapter
During respiratory growth
energy in the most efficient way.
types of respiratory chains,
coli possess several
cytochrome
oxygen it
exclusively
uses
in the presence of
of two aerobic
one
lactate and succinate,
oxidized via
completely
are
oxidases
are
(cytochrome
cytochrome d).
o or
for oxygen and in their turnover rates.
(Km
=
affinity (Km
o,
but
uM)
0.35
resulting
=
3-5
in the
a
rather
nM),
high
generation
terminal oxidase of choice at
active under microaerobic
of
Cytochrome
a
substantially
a
E. coli cells
which
and
can
catalyze
can
(TCA) cycle
acid
quinone pool (43).
one
o
has
low
a
atmospheric
The
of the two terminal
cytochrome
d has
a
high
oxygen
cytochrome
Cytochrome
oxygen levels, whereas
affinity
for oxygen
affinity
lower Vmax relative to
smaller proton motive force.
o
is the
cytochrome
d is
growth (119,120,123).
In the absence of oxygen but in the presence of
acceptor,
non-limiting
These two oxidases differ in their
Vmax. In contrast,
but shows
b to
cytochrome
transferred further via
E.
glucose, pyruvate,
as
tricarboxylic
with concomitant transfer of electrons to the cellular
electrons
Although
respiratory chains, namely
oxidize reduced foodstuffs. Substrates, such
o, to
oxygen, energy is
on
substrate oxidation to the reduction of oxygen.
produced by coupling
amounts of
1: Introduction
produce
a
appropriate
an
set of alternative
alternative electron
respiratory oxidoreductases,
the transfer of electrons to fumarate, nitrate,
dimethyl
sulfoxide
trimethylamine-N-oxide (87,133).
In the
complete
lack of oxygen and in the absence of alternative electron acceptors
the cellular metabolism of E. coli is switched to utilize fermentative
involve redox-balanced dismutation of the carbon source,
These metabolic
changes
substrate
phosphorylation
level
acetate, formate
(H2
and
and
the
formation
CO2), ethanol, lactate,
at the enzyme
the most
energetically
preference
generated
to
synthesis
levels. These
favorable metabolic
nitrate, nitrate
to
of
fermentation
and succinate
The switch between different metabolic modes is
changes
pathways (17).
pathway
mechanisms
is used.
produced
are
7
dramatic
ensure
that
Thus, oxygen is used
fumarate, and fumarate
electron acceptors, which
(17).
accompanied by fairly
regulatory
products:
to
the
in
endogenously
in the fermentative
pathways
Chapter
1: Introduction
(145).
Two sets of
of the
appropriate enzymatic equipment:
synergistically
global transcriptional regulatory proteins
and
in the cellular
antagonistically
adaptation
to
the
on
action of FNR and ArcAB
assisted
other
Figure
by
regulatory molecules,
1. Network of
capitals
and their
italics. All
overlapping
are
the
such
are
given
as
the
can
conditions.
NarLP
to
are
changing
oxygen levels is
(Figure 1) (43).
ovals with the
able to mediate induction and
8
involved
Furthermore, the
given
in
regulatory protein
in
transcriptional regulators. Regulators
as
expression
function both
of certain genes,
expression
adaptation
on
oxygen related
respective modulons
regulators
FNR and ArcAB, which
specific growth
regulatory
ensure
repression
name
of the
of gene
expression.
are
Introduction
Chapter 1:
The
The
global transcriptional regulator
fnr
gene
mutants, which
terminal
are
a
involved in
include
or
is
and
the
the
expression
microaerobic
to
for
0.003
approximately
at the
may
elicits its
FNR
either
act
as
a
of various
transcription
are
oxidase
o
for
alternative
d.
cytochrome
complexes (Table 1).
able to modulate gene
expression
It
in
synergistically or antagonistically.
of
the
1000 molecules per cell.
level in
same
enzymes
terminal
0.01%
-
of
aerobically
and
total
bacterial
Interestingly,
anaerobically
protein,
FNR content
grown E. coli
(146).
Structurally, FNR,
protein
a
of 28 kDa, is similar to
the catabolite repressor
protein,
protein (CRP)
a
different
of E. coli
global regulatory
liganding
of cAMP in CRP,
which includes
allowing
the
an
[Fe-S]
protein
their cognate DNA
protein
consists of
typical
a
an
additional
center and
a
cysteine-rich
slightly
altered
motifs in the
specificity
regulatory
and
a
DNA
regulatory
for DNA
binding
sequences
binding domain,
helix-turn-helix motif, and in which helix E is
9
involved in
are
N-terminal domain in FNR,
to discriminate between FNR and CRP
binding
differences
(128). Major
between FNR and CRP include the lack of amino acids, which
a
is
sequences of FNR activated
cytochrome
the enzymes for
in oxygen levels either
normally
accounts
approximately
cells
and
expression (131). Coding
comprise
changes
corresponding
FNR
as
(125-127).
conditions
is worth to notice that FNR and ArcAB
FNR
thus
growth,
repressor. FNR activates
a
E. coli
fumarate and nitrate
use
in carbon metabolism.
as
anaerobic
encoding
genes
opérons
response to
to
pleiotropic
of E. coli genes. These include genes that
variety
well
as
as
own
respiratory pathways
Repressed
ability
some
narGH]!, frdABCD, dmsABC and cydAB and represses transcription
and its
cyoABCDE,
wide
a
under
activator
as
of
respiration
function
opérons such
opérons
lack the
defects in fumarate and nitrate reduction
transcriptional
of
the isolation of
by
acceptors for supporting anaerobic
global regulator
regulatory
characterized
characteristically
electron
designating
FNR is
initially
was
FNR
thought
sites within
(128).
which is
binding,
The FNR
composed
to lie
across
of
the
Chapter
major
groove of the DNA
acid side-chains
helix F in the
locking
specifically
1: Introduction
have
been
interaction, E209 and S212
(130).
The DNA
two
shown
repeats TTGAT-N4-ATCAA, forming the
binding
by
site
conversion of
FNR into
a
various genes that
are
Contrastingly,
overlaps
a
CRP
activated
the genes that
binding
binding
are
Active FNR, which is in
centers
oxygen, the
site includes two inverted
-35 sequence but have
no
repressed by
change
[4Fe-4S]
transcriptional
FNR have
as
mentioned above
sensing changes
in cellular redox state
might
cysteine
domain, the iron of which is
initiation
FNR
point.
or
is
just upstream
a
[4Fe-4S]
[2Fe-2S] leading
se
Fe-S center.
centers. These
[4Fe-
Upon exposure
to
to the dissociation of
(49, 73,81).
is not the
be the effector in
one
controlling
rich N-terminus forms
but
key regulator
the
part of
a
activity
a
state
redox-sensing
in ferrous state under active anaerobic conditions
most recent results
suggest
a
direct
regulation
of FNR
activity by
(6, 67).
FNR-like
they
an
site, which either
comprises
in oxygen level.
before that oxygen per
of FNR in E. coli. Thus, the
oxygen
a
transcriptional startpoint (+1)
center is converted to
hypothesized
(147). However,
promoters of
Where studied,
(7, 160)
site
the FNR dimer and to the concomitant inactivation of FNR
It has been
FNR-DNA
repeat only, which allows the simple
dimeric form, possess two
a
essential for
are
site directed
for
essential
amino
(50).
domain of FNR
regulatory
Using
some
element and differs from the CRP
core
FNR have
by
the -10 sequence and the
of the -35 sequence
4S]
base in each
be
to
site centered at -39 to -49 relative to the
binding
The
single
a
groove, where
interact with bases in the DNA.
residues
mutagenesis
major
proteins
have been
have been identified both in gram- and
implicated
in the
nitrogen fixation, photosynthesis,
regulation
where
of diverse anaerobic processes, such
and denitrification
10
gram+ bacteria,
(129).
as
Chapter
Table 1.
1: Introduction
Oxygen dependent gene expression
Enzyme
TCA
cycle
Aconitase
mediated
FNR and Arc
Gene
FNR
flcnA
-a)
Arc
related enzymes
(Stat.)
-
acnB
Aconitase
FumaraseA
fumA
FumaraseB
fumB
FumaraseC
fumC
Citrate
by
-
+a)
+
-
Malate DH
-
icd
-
mdh
-
Isocitrate DH
sdhCDAB
Succinate DH
a-ketoglutarate
-
gltA
synthase
DH
Succinyl CoA synthetase
chain
Respiratory
-
-
-
sucAB
-
-
sucCD
-
-
dehydrogenases
Formate DH
fdnGHl
+
Glycerol-3-phosphate DH (anaerobic)
glpACB
+
Glycerol-3-phosphate
Hydrogenase
DH
glpD
(aerbic)
-
hyaA-F
I
L-lactate DH
ild
NADH DH I
nuo
NADH DH II
ndh
Cytochrome
-
+
-
-
cyd
d oxides
-
-
+
-
Terminal oxidases
cyoABCDE
oxidase
Cytochrome
o
Cytochrome
d oxidase
-
-
cydAB
+
-
dmsABC
+
/rdABCD
+
narGH]\
+
anpV
+
NADH-dependent nitrate reductase
nirBDC
+
Formate linked nitrate reductase
nrfA-G
+
focA-pfl
+
+
-
-
DMSO reductase
Fumarate reductase
Nitrate reductase
Nitrate reductase
(periplasmic)
+
Others
Formate
transport and pyruvate-formate lyase
Pyruvate
DH
complex
and
pdhR-aceEF-lpd
regulator
VHb
vhb
+
FHP
jhp
+
a) +
denotes
The
as
transcriptional activation,
expression
the
hmp
whereas
of various bacterial
genes from Ralstonia
-
denotes
repression
hemoglobin
eutropha,
E.
11
and
of the
respective genes.
flavohemoglobin
genes, such
coli, Vitreoscilla and Bacillus subtilis have
Introduction
Chapter 1:
been shown to be controlled
regulation, although
of these
expression
other
in oxygen levels via FNR mediated
by changes
regulatory proteins
diversely regulated
genes
involved in the
are
regulation
of
(18, 74,85, 111).
ArcAB modulon
The ArcAB
system
was
characterized
isolated E. coli mutants, which
by
unable to repress
were
(64, 66, 86). They
Lin and coworkers
whole set of aerobic
a
functions, including TCA cycle, the glyoxylate cycle, fatty acid oxidation enzymes
and
cytochrome
designating
The DNA
The
o.
corresponding
their lack of aerobic
consensus
respiration
were
are
regulated by
for
sequence
ArcA and
binding
ArcA
named arcA and arcB,
control.
multiple alignments
site of ArcA has been defined from
binding
promoter sequences that
The
mutant genes
by
footprinting
DNA
base
10
includes
of
assays.
pairs
(5'-
[A/T]GTTAAGGA[A/T]-3') (91).
ArcAB is
sensor
two
a
ArcB, detecting
a
activity
is able to
ArcB has both kinase and
auto-phosphorylate
ArcB
stimulated, ArcB undergoes auto-phosphorylation
group
is
probably
concomitantly
a
phosphoryl
Asp576
and
conformational
phosphorylated
during
membrane bound
intramolecularly
change.
This
at
the
next
ArcA may function either
as a
well
His292.
transferred
change
round
phosphatase
as
allows
group from His292 to ArcA instead to the
residue
a
switch from aerobic to anaerobic metabolism and the
cognate regulatory protein ArcA.
The kinase
consists of
component regulatory system, which
of
to
ArcB
as
activities.
ArcA. When
The
phosphoryl
Asp576, causing
to
transfer
the
already phosphorylated
auto-phosphorylation.
transcriptional
activator
or
When
repressor
(62, 65).
The stimulus for ArcB
instead it has been
the
phosphorylation
suggested
cytosol regulate
does not appear to be oxygen itself but
that either the state of
the rate of
respiration
phoshorylation (63, 86).
12
or
the redox state of
It has been
suggested
that
Chapter
the
phosphatase activity
activity. Metabolites,
during
accumulate
groups to ArcA
key player
lactate,
as
growth,
regulation
in the
pyruvate,
inhibit the intrinsic
phosphatase activity
and
acetate
of the ArcAB
are
proposed
signaling
and
prokaryotes
to
phosphatase activity
a
where
are
widely
they
suggests that the
distributed in sensory
monitor
changes
light,
in
PAS domain of ArcB
decrease in O2 tension. It is therefore
electron flux
phosphoryl
superfamily.
proteins
likely
PAS
from
redox state and
might respond
reduced intermediate that accumulates when electron flow to O2 is
following
of
phosphorylation
leads to the irreversible
be member of the PAS domain
domains that
eukaryotes,
This
(139).
which
NADH,
(62).
ArcB has been
oxygen
a
of AcrB and therefore, to the intermolecular transfer of
Asp576
domains
such
anaerobic
ArcB. The lack of the
of
of ArcB is
1: Introduction
to
a
interrupted
that ArcB may monitor the
oxygen itself
trough the respiratory chain rather than sensing
(91).
Oxidative and nitrosative stress in E. coli
Oxidative and nitrosative stress
are a common
exposure of the cell to reactive oxygen and
biomolecules
damage
Organisms
lifetime.
or
such
Endogenously,
common
processes,
mechanisms such
proteins,
endogenously
face both
denitrifying
as
as
and
phenomenon
are
nitrogen intermediates,
nucleic
acids
and
exogenously produced
metabolic processes, such
produce
and
as
or
which
cell
stress
by
can
the
both
membranes.
during
their
respiratory activity
these stress factors; exogenous
attack of the immune system
caused
sources
plant pathogen
include
response
against invading cells.
It is
now
endogenously
in the
is
generated
intermediate in the reduction of nitrate to
dinitrogen
well established that the radical
as an
denitrifying
lO'9 M and IO-7 M
obligate
nitrogen
monoxide
(NO)
bacteria. The concentration of NO is estimated to vary between
(47).
The formation of NO is
13
catalyzed by
nitrite
reductases, such
1: Introduction
Chapter
as
copper,
NO is
In
cdl reductases, where nitrite is reduced to NO; the
cytochrome
reduced to nitrous oxide
subsequently
exponentially growing
autooxidation of
dehydrogenase
respiratory
10"6 M,
bacteria both H2O2 and
II is the
primary
Steady-state
respectively (46,48,59).
concentration of O2
Both levels
example, pathogenic
various ROS and RNS when
utilized
are
are
species (ROS)
formation of reactive oxygen
products
they
by phagocytes
and reactive
are
captured by
to disarm
nitrogen species (RNS)
bombarded
the
expressed
damage.
threatening
these
enzyme
machinery
Under normal
radicals
produced
and
exposure of cells to
a
a
phagocytes.
invading bacteria
bacterial
cells
that detoxifies the reactive
growth
the
radicals
conditions
a
defense
cellular
are
under the
are
collection of
The arsenal of
include O2
,
NO,
species
reactive
on
is
their
(134).
common
possess
species
constitutively
and
repairs
the
balance between the amount of
mechanisms
boost of radicals results in the
defense enzymes, which
regulatory proteins
by
bacteriocidal, but their impact is strongly dependent
or even
overcome
toxicity (134).
if the rates of intracellular
inadequate,
ultimate concentrations inside of the bacteria
To
the
10'10 M and IO-7-
are
still below the level of
bacteria
are
and H2O2
HOC1, H2O2, HO", HOONO and RSNO (27). Each of these
bacteriostatic
generated by
are
site of electron transfer to oxygen in the aerobic
The constitutive defense mechanisms
toxic
superoxide
(47).
components of the respiratory chain (46, 59). The flavin of NADH
chain.
enhanced. For
nitric oxide reductase
by
produced
rapid
global
exists.
In
induction of
addition,
adaptive
control of SoxRS and
OxyR
in E. coli.
SoxRS modulon
The SoxRS
common
regulon comprises
the induction
a
variety
by superoxide
of defense related enzymes, which have in
radicals and NO. SoxRS is
transcription factor,
regulatory system.
SoxR is
of SoxS. SoxR is
homodimer of 17 kDa per
a
a
latent
14
a
two-component
which controls the
polypeptide,
which contain
activity
a
helix-
Chapter
turn-helix DNA
redox-sensitive
the
protein.
superoxide
NADPH
binding
motif in the N-terminal domain. At its C-terminus,
[2Fe-2S]1+
It is not
cluster is located
yet clear if the
and NO. Indirect
pool;
SoxR
means
Upon
protein directly
the size of it in turn would be
auto-regulation
oxidation
conformational
of
cluster
change,
which
to
of
indirectly responds
to
depletion
of the cellular
the SoxS
responsive zwf
regulated by
The latter mechanism would represent
mechanism of SoxRS
the
or
the
an
activity (162).
[2Fe-2S]2+,
enables
a
activity
which controls the
(55,159),
of activation include
(glucose-6-phophate dehydrogenase).
attractive
1: Introduction
the
protein undergoes
SoxR
protein
to
regulatory
the
activate
a
sequence of soxS gene. The up to 100-fold increase in SoxS level in turn mediates
thereafter the
superoxide
the
dismutase
superoxide
(acnA)
in E.
(sodA),
the DNA
radical resistant
confer resistance
reducing
against
many different
drugs
(nfo),
(fumC)
and
and aconitase
regulon
well
as
as
has been found to
organic
solvents and
nitrogen species (99,104).
The
expression of many H2O2 inducible
modulon
transcription
peroxide (15).
factor
in
Active
helix-turn-helix DNA
E.
OxyR
coli.
forms
binding
conserved bases. The
binding
a
are
by
H2O2
formed and the
are
regulated by
hypersensitive
the
to
OxyR
hydrogen
tetramer in vivo with subunits of 34 kDa. The
motif is located at the N-terminus. The
pair
sequences, which
sequence of
of various E. coli promoters
is activated
activities is
OxyR-mutants
sequence includes four sets of 4 base
bridges
manganese
endonuclease IV
of fumarase
power of the cell. SoxRS
oxyR
OxyR
as
glucose-6-phosphate dehydrogenase (zwf),
The
region
such
proteins,
repair enzyme
isoenzymes
coli. SoxS also activates
which increases the
reactive
of the various defense
expression
OxyR
is
are
spanning
binding
separated by
7
non-
between -35 and -80
(140).
through
protein
oxidation.
Upon
oxidation of
OxyR,
disulfide
is in its active conformation. Mutation of either
15
Chapter
of the two conserved
abolishes the
activated
ability
only
for
sense
finite
a
inactivation
of
glutaredoxin
1 that is itself
inactivation
of
concentration
required
OxyR
(53).
after
by
as
depletion of
The members of
glutathione
hydrogen peroxide,
OxyR regulated
reactive
OxyR
that the
bridges by
(gorA).
In
of
cysteine
(53).
similar
are
(4).
Activation of
OxyR by
by
oxidation
residue and not
OxyR by
are
nitrosative stress
synergistic
and the
the cellular metabolism, for
on
pool.
include catalase
oxyR
peroxide
hydrogen
vivo is 5 uM
OxyR in
the effects of ROS and RNS
the cellular GSH
OxyR regulon
reductase
proposed
disulfide
the
minimal
that the activation of
effects of ROS and RNS exposure
the
The
nitrosative stress
by S-nitrosylation
effects is reasonable inasmuch
example
of
reduction
oxidize
completely
(53) suggest
Hausladen et al.
the
auto-mediated.
is activated
occurs
of time in vivo. It has been
part of the OxyR response regulon (117,161). Thus, the
is
to
remains oxidized and
hydrogen peroxide. OxyR
period
occurs
OxyR
Furthermore, OxyR
nitrosative stress
residues involved in disulfide bond formation
cysteine
to
1: Introduction
(katG), glutaredoxin
strains 20 to 30
but theses strains have increased
activities mediate resistance
proteins
are
spontaneous
1
(grxA),
and
still inducible
by
mutation rates.
against HOC1, organic
solvents and
nitrogen species (134).
and SoxRS
independent functions against nitrosative
and
oxidative stress
Besides genes that
are
the above-described
regulated
in response to nitrosative and oxidative stress
transcriptional regulatory proteins,
evidence that other cellular
components
towards ROS and RNS, which
are
are
there exists accumulated
also involved in the defense response
regulated independently
16
by
of SoxRS and
OxyR.
Chapter
Role of microbial
flavohemoglobins
1: Introduction
in the defense
against
nitrosative and
oxidative stress
from several bacteria and
Flavohemoglobin proteins
major
yeasts have been shown
to be
contributors to the detoxification of NO under aerobic conditions and to be
essential for resistance
against
of E. coli has been shown to be
flavohemoglobin
but to be controlled
by
(20, 40, 112).
nitrosative stress
MetR
(95, 111).
independent
expression of
The
of SoxRS
regulation
flavohemoglobins
The role of
in the
resistance to nitrosative stress is discussed below.
The
knowledge
in its
potential
reported,
role
as
flavohemoglobin of
NO reduction
Recent
by
flavohemoglobins
the involvement of these
infancy. Although
stress has been
A
the interaction of
on
it is not
amplifier
E. coli
yet clear what role these proteins play
of
oxidative
(36, 37)
Gardner and coworkers
pathway
in E.
conditions. The Operon includes three genes
and
gene cluster
encodes
a
been
proposed
for the
ygaD
encode
a
as
showed the existence of
coli, which is
ygaAKD. YgaA
flavorubredoxin
oxidoreductase, respectively (36, 37). Gardner
YgaA
has
stress
in bacteria.
(112).
additional NO' detoxification
ygaAKD
in response to oxidative
proteins
«orRVW modulon
findings by
regulator; ygaK
and oxidative stress is still
et al.
(RB)
an
active under anaerobic
encodes
and
a
(37) proposed
a
transcription
NADH:flavoRB
to
designate
the
the norRVW modulon for NO reduction and detoxification.
protein bearing
euthropha transcription regulators
42%
identity
with the NO-modulated Ralstonia
NorRl and NorR2
also encountered in Vibrio cholera and Pseudomonas
17
(110).
NorR
homologous
aeruginosa (54,135).
are
Chapter
Hemoglobins:
Hemoglobins
are
are
encountered in all five
encountered
characteristic of all
a
overview
an
kingdoms
Hb's
organisms. Generally,
of
classified into vertebrate and nonvertebrate Hb's. The latter group also includes
hemoglobins
the
1: Introduction
highly
residues
variable
are
and His at
protein
or even
is their
of
ability
reversibly
structure, the classical Hb fold. This
six
intervening loops.
a-helical
eight
to
structures,
moiety
within
a
sources
reveals
key
a
are
CD1
position
reveals
hemoglobins
highly
which
The 3-on-3 helix structure forms
which is able to bind the heme
Although
bind oxygen.
far: Phe at
so
structures of
protein
common
amino acid sequence, two
primary
encountered
proteins
of
to
The
protozoa.
hemoglobins from various
almost random
Analysis
F8.
position
and
fungi,
sequences of
conserved among all
typical tertiary
consists
hemoglobins
of the
alignment
plants,
in
a
conserved structure
connected
by
short
assembly,
sandwich-like
cavity surrounded by hydrophobic
residues.
Phylogenetic
common
trees based
and
Vertebrate
serve
oxygen
donate oxygen to the
discovered in
to deliver
nerve
encountered in
much
freely
higher
variability
or are
expressed
respiring
which
proposed
occur
widely
to
and
x
circulating
This
Neuroglobin
represent
possibly
in
a
has
distinct
to
an
where
they
they
alternative Hb has been
neuroglobin has
a
where
unique
been
proposed
exon-intron structure
protein family (13).
from monomeric to multisubunit structures and
dissolved in various
primary
their
much
body
and
18
of
cytoplasm
fluids. Nonvertebrate Hb's
tertiary
broader
a
primitive
a
(myoglobin)
different anatomical sites either in
indicate
is
body fluids,
in muscles tissues
mouse.
point
sequences
109 years ago.
Very recently,
cells.
man
variation in their
may
ancestor,
encountered
tissues of
Nonvertebrate Hb's
or
are
oxygen to brain tissue.
and therefore it is
tissues
globin
developed approximately 3.5
hemoglobins
to deliver
present-day globin
the known
ancient
quite
archaebacterium that
on
structures. This
defined
are
specific
display
high degree
adaptations
to
a
of
specific
Chapter
functions
compared
vertebrate Hb is not
to
as
1: Introduction
their vertebrate
complete
in the
as
Over the last dozen years, monomeric
symbiont-containing leguminous
of
N-terminal
domain
globin
(collectively
domain and
called
domain
globins
have been found in
nonleguminous plants, algae,
cyanobacteria.
flavohemoglobins)
and
Chimeric Hb's
number
a
of
consisting
C-terminal redox-active
adjacent
an
non¬
of vertebrate Hb.
case
single
from bacteria to
prokaryotes, ranging
an
and
about
knowledge
The
homologs.
protein
have been found in bacteria and
yeast. Furthermore, truncated Hb's have been identified
in
bacteria and
plants,
cyanobacteria (98,116,150).
Bacterial
hemoglobins
Identification of
primary
Bacterial
and
hemoglobins
microorganisms.
genes that
due to
hemoglobins
high
flavohemoglobins
flavohemoglobin proteins
amino acid
homologies
proteins,
hemoglobin
indoleamine
Vitreoscilla
bacterial
and their
by
and
flavohemoglobins
of genes whose existence is
to known
proteins
is
designated
shortened
hemoglobins
protein
given
as
a
predicted
in Table 2. Besides
truncated
sequence of
suggested
are
a
not to
hemoglobins
approximately
originate
different gene,
from
namely
an
2,3-dioxygenäse (136).
proteins.
Vitreoscilla, which lives
primary protein
weight
the amino acid
VHb
in
is the best-characterized member of the group of
is
expressed
under
conditions
oxygen-limited
in
stagnant ponds and decaying plant material (153). The
sequence and the DNA sequence of VHb have been
The gene
molecular
a
have been identified in
hemoglobins
but to have evolved from
hemoglobin (VHb)
Hb
or
third group,
a
110-120 amino acids. Truncated
ancestral
of
A summary
exists, and it is characterized
149).
and
isolated and characterized
are
these two groups of
77,
flavohemoglobins
sequences
of
variety
and
encoding
VHb is 441
of 15.7 kDa
as
bp long,
determined
composition (149). Initially
19
which results in
experimentally
called
cytochrome
a
reported (25,
protein
of
a
and calculated from
o,
VHb
was
believed
Chapter
to
be
soluble terminal oxidase in
a
purifying VHb,
Table 2.
an
1: Introduction
Vitreoscilla
NADH metHb reductase
also
at the Gene
bank,
or
of
genomic
Organism
Campylobacter jejuni
are
currently
potential
genes
Gene
Gene bank
Length
(bp)
Reference
chb
AL139079
423
(8)
Cp-/ib
AB028630
435
(33)
vhb
M30794
441
(25,77)
NCTC11168
perfnngens hyp27
which
sequences which allow the identification of
globin
Vitreoscilla sp.
AE000678.1
Aquifex aeohcus
NC.000918.1
420
Bacillus halodurans C-125
hmpBh
AB024563
1236
(33)
Bacillus subtilis
hmpBs
D78189
1200
(85)
Deinococcus. radtodurans
hmpDr
AE001863
1212
(8)
Escherichia coli MG1655
hmp
X58872
1191
(148)
hmpX
X75893
1188
(84)
hmpPa
AE004695
1182
(8)
flip
X74334
1212
(18)
NCL002758.1
1146
AP003129.2
1146
Erwinia
168trpC2
chrysanthemi
Pseudomonas, aeruginosa PAOl
Ralstonia
eutropha
Staphylococcus,
aureus
MU50
Staphylococcus aureus N315
Streptomyces
coehcolor A3
hmpA2
AL354616.1
1212
(33)
Streptomyces
coehcolor A3
hmpA
AL158061
1197
(33)
Streptomyces
coehcolor A3 cosmid Jll
AL109949.1
1308
hmpSi
AL627275
1191
(8)
hmpStm
AF020388
1191
(19,20)
AE008816.1
1191
(19,20)
NC_003037
1212
(33)
AE004358.1
1185
U09005.1
1185
Salmonella entenca
subsp. Typhi
Salmonella
typhimunum
Salmonella
typhimunum
LT2
hmpSm
Sinorhizobmm meliloti
Vibrio cholerae
Vibrio
parahamolyticus
Xylella fastidwsa
Yersinia
AE003859.1
9a5c
NC_0024881
pestis OC92
AJ4141541
VHb is encountered in vivo
as a
share this property of VHb.
summarized
in
this
work
(152).
work
on
Further
20
1194
1191
it is not known if other
dimer, but
Early
when
copurified (45,154).
Compilation of bacterial hemoglobin and flavohemoglobin genes,
deposited
Clostridium
was
(153, 154). Interestingly,
hemoglobins
VHb has been reviewed and is not
hemoglobin proteins,
which include
Chapter
hemoglobins
from
and
Campylobacter jejuni
isolated and characterized in
The
1: Introduction
strictly respiratory,
our
coding
a
-
a
denitrifying, Gram-negative
flavohemoprotein
sequence of this
protein
is
or
the amino-terminal
on
determined
previously by
plasmid
monomeric
polypeptide
R.
eutropha,
form
of
bacterial
alternative
determined
antibodies and
Riggs (163).
of 403 amino acids
called FHP
(158)
sequence of
polypeptide
Zhu and
an
bacterium
flavohemoglobin
megaplasmid library by using FHP-specific
based
been
laboratory (8,33).
formerly Alcaligenes eutrophus, synthesizes
hemoglobins
have
perfringens,
Clostridium
The
having
a
a
flip
and
was
(118).
The
isolated from
oligonucleotide probes
yeast flavohemoglobin,
gene encodes
molecular
weight
a
soluble
of 44 kDa
(18).
FHP
M
LTQKTKDIVKATAPVLAEHGYDIIKCFYQRMFEAHPELK-NVFNMAHQEQGQQQQALARAVYAYAENIEDPNSLM
75
HmpEc
M
LDAQTIATVKATIPLLVETGPKLTAHFYDRMFTHNPELK-EIFNMSNQRNGDQREALFNAIAAYASNIENLPALL
75
HmpStm
M
LDAQTIATVKATIPLLVETGPKLTAHFYDRMFTHNPELK-EIFNMSNQRNGDQREALFNAIAAYASNIENLPALL
75
HmpSt
M
LDAQTIATVKATIPLLVETGPKLTAHFYDRMFTHNPELK-EIFNMSNQRNGDQREALFNAIAAYASNIENLPALL
75
HmpX
M
LDQQTIATIKSTIPLLAETGPALTAHFYQRMFHHNPELK-DIFNMSNQRNGDQREALFNAICAYATHIENLPALL
75
HmpBs
M
LDNKTIEIIKSTVPVLQQHGETITGRFYDRMFQDHPELL-NIFNQTNQKKKTQRTALANAVIAAAANIDQLGNII
75
HmpBh
MSTATLSQETKQIVKATVPILAEHGEAITKHFYKRMFSHHPELL-NIFNQTHQKQGRQPQALANSIYAAAEHIDNLEAIL 79
HmpDr
M
LTPEQKAIVKATVPALEAHGETITRTFYASMFAAHPELL-NIFNPANQQTGKQARSLAASVLAYAAHIDHPEALG 75
HmpPa
M
LSNAQRALIKATVPLLETGGEALITHFYRTMLGEYPEVR-PLFNQAHQASGDQPRALANGVLMYARHIDQLQELG
HmpSc
M
LSSQSAQIVRDTLPTVGASLGTITDLFYRRMFEERPELLRDLFNRANQASGVQREALAGAVAAFATALVKHPDER 76
CHb
M
TKEQIQIIKDCVPILQKNGEDLTNEFYKIMFNDYPEVK-PMFNMEKQISGEQPKALAMAILMAAKNIENLENMR
74
Cp-Hb
M
LDQKTIDIIKSTVPVLKSNGLEITKTFYKNMFEQNPEVK-PLFNMNKQESEEQPKALAMAILAVAQNIDNLEAIK
75
VHb
M
LDQQTINIIKATVPVLKEHGVTITTTFYKNLFAKHPEVR-PLFDMGRQESLEQPKALAMTVLAAAQNIENLPAIL
75
75
152
FHP
A-
-VLKNIANKHASLGVKPEQYPIVGEHLLAAIKEVLGN-AATDDIISAWAQAYGNLADVLMGMESELYERSAEQPGGWK
HmpEc
P-
-AVEKIAQKHTSFQIKPEQYNIVGEHLLATLDEMFSP
GQEVLDAWGKAYGVLANVFINREAEIYNENASKAGGWE 15 0
HmpStm
P-
-AVEKIAQKHTSFQIKPEQYNIVGTHLLATLDEMFNP
GQEVLDAWGKAYGVLANVFIHREAEIYHENASKDGGWE 150
HmpSt
P-
-AVEKIAQKHTSFQIKPEQYNIVGTHLLATLDEMFNP
GQEVLDAWGKAYGVLANVFIHREAEIYHENASKDGGWE 150
HmpX
P-
-AVERIAQKHASFNIQPEQYQIVGTHLLATLEEMFQP
GQAVLDAWGKRYGVLANVFIQRESDIYQQSAGQNGGWH 15 0
HmpBs
P-
-WKQIGHKHRSIGIKPEHYPIVGKYLLIAIKDVLGD-AATPDIMQAWEKAYGVIADAFIGIEKDMYEQAEEQAGGWK
HmpBh
P-
-WSRIAHKHRSLNIKPEQYPIVGENLLAAMREVLGD-AASDDVLEAWREAYELIADVFIQVEKKMYEEASQAPGGWR 156
HmpDr
G-
-MVGRIAHKHVSLEVLPEHYPIVGQYLLGAIAGVLGD-AAKPEILDAWAAAYGELADLMIGIEKGMYDAGAGQPGGWR
152
HmpPa
P-
LVAKWNKHVSLQVLPEHYPIVGTCLLRAIREVLGEQIATDEVLEAWGAAYQQLADLLIEAEESVYAASAQADGGWR
153
HmpSc
PDAVLGRIANKHASLGITSDQYTLVGRHLLAAVAEVLGD-AVTPAVAAÄWDEVYWLMANALIAMEARLYARSDVEDG-
CHb
S
Cp-Hb
P--WNRIGVIHCNAKVQPEHYPIVGKHLLGAIKEVLGD-GATEDIINAWAKTYGVIAEVFINNEKEMYASR
144
VHb
P--AVKKIAVKHCQAGVAAAHYPIVGQELLGAIKEVLGD-AATDDILDAWGKAYGVIADVFIQVEADLYAQAVE
146
-
-
FVDKVAITHVNLGVKEEHYPIVGACLLKAIKNLLN-
-
-
PDEATLKAWEVAYGKIAKFYIDIEKKLYDK
152
153
140
FHP
GWRTFVIREKRPESDVITSFILEPADGGPWNFEPGQYTSVAIDVPALGLQQIRQYSLSDMPNGRTYRISVKREGG-G-P 230
HmpEc
GTRDFRIVAKTPRSALITSFELEPVDGGAVAEYRPGQYLGVWLKPEGFPHQEIRQYSLTRKPDGKGYRIAVKREEG
226
HmpStm
GTRPFRIVAKTPRSALITSFEFEPVDGGTVAEYRPGQYLGVWLKPEGFAHQVFRQYSLTRKPDGKGYRIAVKREDG
226
HmpSt
GTRPFRIVAKTPRSALITSFEFEPVDGGTVAEYRPGQYLGVWLKPEGFAHQEIRQYSLTRKPDGKGYRIAVKREDG
226
HmpX
GIRPFRIVAKQPQSSLITSFTLEPVDGGPIAAFRPGQYLAVYIRDKRFEYQEIRQYSLTNEPNGRYYRIAVKRETM
226
HmpBs
EYKPFVIAKKERESKEITSFYLKPEDSKPLPEFQAGQYISIKVRIPDSEYTHIRQYSLSDMPGKDYYRISVKKD
226
HmpBh
EFRSFWEKKQRESATITSFYLKPEDGKALASYKPGQYITVKVTIPGHEHTHMRQYSLSDAPEKGYYRITVKREEGDG-D 235
HmpDr
DFRPFRVARKVAESRVITSFVLEPVGGGALPAYQPGQYLSLKVKVPGQERWQIRQYSLSDAPSPDHYRISVKREGG
228
HmpPa
GVRRFRVARKQAESEEITSFYLEPVDGQPLLAFQPGQYIGLRLDIDGEE--VRRNYSLSAASNGREYRISVKREAG-
227
HmpSc
VWQSMEIVERHEETPDTASFVLRPADGSPTRPFVPGQYVSVRAELPDGAH-QIRQYSLSSAPGGGTWRFTVKRERSLDGQ 232
21
Chapter 1:
Introduction
QPPGYVSNLLHDHVNVGDQVKLAAPYGSFHIDVDAKTPIVLISGGVGLTPMVSMLK-VALQAPPRQWFVHGARNSAVHA
309
HmpEc
GQVSNWLHNHANVGDWKLVAPAGDFFMAVADDTPVTLISAGVGQTPMLAMLDTLAKAGHTAQVNWFHAAENGDVHA
303
HmpStm
GQVSNWLHHHASVGDWHLAAPAGDFFMNVAADTPVSLISAGVGQTPMLAMLDTLAKEQHTAQVNWFHAAENGDVHA
303
HmpSt
GQVSNWLHHHANVGDWHLAAPAGDFFMNVAADTPVSLISAGVGQTPMLAMLDTLAKEQHTAQVNWFHAAENGDVHA
303
HmpX
GSVSGYLHDVAREGDVIELAAPHGDFYLEVTPETPVALISAGVGQTPMLSMLHSLKNQQHQADIFWLHAAENTEVHA
303
HmpBs
GWSSYLHDGLQEGDSIEISAPAGDFVLDHASQKDLVLISAGVGITPMISMLKTSVSKQPERQILFIHAAKNSEYHA
303
HmpBh
LPPGIVSNYLHQHIHEGDVLEITAPAGDFTLQEEGERPIVFISGGVGITPLMSMFNTLMQRGVKREVIFIHAAINGFYHA
315
HmpDr
304
HmpPa
GLVSEYLHGAVQEGDELLVHVPAGDFVLQQ-SERPWLISAGVGITPMLAMVQTLAQAGSQRPVTFIHAAQNGSVHA
GRVSNYLHDRVAEGDELDLFPPAGDFVLRD-SDKPLVLITAGVGITPALAMLQEALPQA--RPIRFIHCARHGGVHA
HmpSc
VPDGEVSTWLHTHARPGDVLRVSLPFGDLLLPE-GDGPLLLASAGIGVTPMLAMLEHLATAAPDRPVTWHADRSPALHA
311
FHP
MRDRLREAAKTYENLDLFVFYDQPL-PEDVQGRDYDYPGLVDVKQIEKSILLP-DADYYICGPIPFMRMQHDALKNLGIH
3 87
HmpEc
3 81
HmpStm
FADEVKELGQSLPRFTAHTWYRQPS-EADRAKGQFDSEGLMDLSKLEGAFSDP-TMQFYLCGPVGFMQFTAKQIiVDLGVK
FADEVSELGRTLPRFTAHTWYREPT-EADRAQRLFDSEGLMDLSKLEAAISDP-AMQFYLCGPVGFMQFAAKQLVSLGVN
HmpSt
FADEVSELGRTLPRFTAHTWYREPT-EADRAQRVFDSEGLMDLSKLEAAISDP-AMQFYLCGPVGFMQFAAKQLVSLGVN
3 81
HmpX
FADEIADVAATLPQLQSYVWYREASSEAARSAHAFH- -GLMALKDLPTPLPMT-NLHCYLCGPVAFMQFAARQLLELGIT
380
HmpBs
LRHEVEEAAKHS-AVKTAFVYREPT-EEDRAGDLHFHEGQIDQQFLKELIANT-DADYYICGSPSFITAMHKLVSELGSA
380
HmpBh
393
HmpPa
MHDHLAQTASQQENVHYAVCYERPT-PGDRMNPFMKKEGFIDESFLRSILHDR-EADFYFCGPVPFMKTIAQILKDWDVP
FRDDVARLTHEYPHFRKWFYDEAG-PDDQLGTHHDVAGRLSLDAVRGALPAG-EAEFYYCGPAGFAGAVEAILDDLQVP
RAGDSADAEGLLSREKLADWLPQERDLDAYFLGPRPFMAQVKRHLADLGVP
FRDWIEDVSAQHEQVEHFFCYSEP
HmpSc
HRLELTALVERLPHASLHLWYEDTADHPDASADHVN-EGWADLTGLSPVPGTT
AFLCGPLSFMKAVRTDLLAHGLS
386
FHP
HmpDr
FHP
EARIHYEVFGPDLFAE
403
HmpEc
QENIHYECFGPHKVL
396
HmpStm
NENIHYECFGPHKVL
396
HmpSt
NENIHYECFGPHKVL
396
HmpX
ESQIHYECFGPHKVI
395
HmpBs
PESIHYELFGPQLSLAQSV--
399
HmpBh
EQQVHYEFFGPAGTLASS
411
HmpDr
AERRFTETFGPSQS FAPVILG 403
HmpPa
SQQCHYEFFGPAAALDA
393
HmpSc
PRAIHYEVFGPDLWLSK
403
Figure
recently
2.
amino acid
Multiple
been studied. VHb
alignment
of
and
flavohemoproteins
(Vitreoscilla, (25, 77), HmpEc (E. coli, (148),
381
382
376
which have
hemoglobins,
FHP
301
(R. eutropha, (18)), HmpX
(E. chrysanthemi, (35)), HmpBs (B. subtilis, (85)), HmpStm (S. typhimurium, (19, 20)) CHb, HmpPa,
and
HmpDr
HmpSt (C. jejuni,
D.
radiodurans, S. typhi, (8)), HmpSc, HmpBh, and Cp-Hb (S.
coeolicolor, B. halodurans, C. perfringens, (33)). Throughout all sequences conserved amino acids
are
highlighted.
Flavohemoprotein
dihydropteridine
from E. coli
(HMP)
high
amino acid
typically
domain at the N-terminus with
domain
chrysanthemi,
B.
at
the
a
The molecular
weight
encountered in
identify
to
of HMP is 44 kDa and
characteristic
C-terminus.
flavohemoproteins;
ability
to abduct CO and
Previously, flavohemoglobins
from
a
a
same
globin
redoxErwinia
subtilis, Salmonella typhimurium and Mycobacterium tuberculosis have
been isolated and characterized at the level of DNA and
Further
attempt
with FHP. Both HMP and FHP share the
similarity
structural characteristics
active
an
activities and it is up to date the best characterized member of
flavohemoglobin protein family (148).
shows
has been isolated in
flavohemoglobin proteins
have been
22
recently
protein (19, 20, 35, 57, 85).
isolated from Deinococcus
Chapter
Pseudomonas
radiodurans,
Streptomyces coelicolor,
sequences of
Salmonella
approximately
approximately
The
aeruginosa,
flavohemoglobins
of
proteins
alignment
1: Introduction
Klebsiella
typhi (Table 2) (8, 33).
is
approximately
400
length
of the
coding
basepairs long, resulting
1200
acids
amino
The
halodurans,
Bacillus
pneumoniae,
and
a
weight
molecular
in
of
44kDa.
of the
residues in both the
protein
sequences reveals the
occurrence
of several conserved
domain and in the redox-active domain
hemoglobin
(Figure 2).
The role of these conserved residues is discussed below.
Structure and biochemical
properties
Structure of VHb and FHP
The three dimensional
ray
structures of FHP and VHb have been resolved
(3-D)
crystallography (30, 31,137,138).
The 3-D structures of the
of FHP and VHb conform to the well-known
embedded in
a
rigid binding
between the
hydrophobic
crevice formed
polypeptide
predominantly by hydrophobic
covalently
by
globin
hemoglobin
by
X-
domain
fold. The heme molecule is
the helices B, C, E, F, G and H. A
chain and the
porphyrin ring
side chains towards the
the heme in both FHP and VHb. No
is
provided
side. His85 binds
proximal
protein-heme
interactions
are
detected at the distal side of the heme molecule. A clear difference of these
two
is the lack of helix D, i.e.
the
structures
residues
relative to mammalian
connecting
helices C and E do not
The observed disorder in the CE
of the
globin
would
are
highly
FHP is the
F. The
adopt
region might
the usual a-helical conformation.
indicate
a
potential
site of interaction
domain of FHP with the FAD of its reductase domain. In
provide
reductase.
hemoglobins
a
potential
contact site of VHb with the NADH
(9, 30,138).
Overall the structures of VHb and the
similar
A
(9).
displacement
heme-binding
striking
globin
difference between VHb and the
of helix E in FHP,
crevice is therefore
opening
enlarged
23
the
thus
angle
analogy,
this
methemoglobin
domain of FHP
globin
domain of
between helix E and
providing
more
space at the
Chapter
ligand-binding
(30). Indeed,
site
hydrophobic crevice,
The
a
1: Introduction
but the function of this
ligand
is not known
quaternary assembly of homodimeric VHb, which has
before within the
globin family,
F and H of both subunits and
is based
on a
essentially
hemoglobins,
In contrast to the classical
active domain with
potential binding
FHP contains
sites for
form
group of
a
functional and structural
reductase domain
NADH
can
respectively
helix
one
proteins,
on
a
adopts
which do not share any
a
six-stranded
FNR. The
each side. The FAD group binds
and
a
antiparallel
parallel ß-sheet
five-stranded
a
FNR-like
binding (residuesl 53-258)
domain. The former consists of
the latter is built up of
(138).
and FAD, which
(FNR)-like proteins (72).
FAD
a
helices
C-terminal-located redox-
a
global regulatory protein
with the
be divided into
binding (266-403)
ß-barrel, whereas
properties
by
distance of 34 A
a
NAD(P)H
related
structurally
observed
not been
der Waals contacts among
van
approach to
fold similar to ferredoxin NADP+ reductase
proteins
(105).
molecular interface defined
involves
the two subunits. The two heme-iron atoms
two
has been found to bind the
phospholipid
by
flanked
non-covalently
in
an
extended conformation to all three domains of FHP. The heme and FAD molecules
o
approach
each other to
FAD to heme
moiety
a
in
amino acid side chains
spatial arrangement
minimal distance of 6.3 A
a
predominantly polar
(30).
FHP
displays
of FAD and NADH
a
enabling
electron transfer from
environment
large
binding
provided by
several
inter domain cleft. The relative
domains within FNR-like
allows variance. A clear characterization of NADH
binding
to FHP
proteins
has not been
shown.
In
general, hemoglobins
are
and
share
invariant in all known sequences. The role of
known for
some
the sequences
standard
characterized
(Figure 2).
globin fold)
a
stability.
In
proteins
Around
Trpl22(H7).
all known
or can
a
some
number of residues, which
of the conserved residues is
be derived from the
homologies
Phe43(CDl) (topological position according
hydrophobic
cluster is centered around
and
flavohemoglobins
cluster is formed. A further
These two clusters
globins Phe43(CDl)
24
and
are
the
hydrophobic
essential for
His85(F8)
to
of
are
folding
invariant.
Introduction
Chapter 1:
His85(F8), Tyr95(G5)
interaction of
atom and
c
Glul37(H23)
Fe-His-Glu-Tyr
peroxidase.
transform
the
protein
These
bacterial
(30). Tyr29(B10)
highly
binding
conserved:
proteins
thought
is
heme, Gln207 is replaced by Asn
a
in
chains
originate
part of
electrochemical
a
to stabilize the
potential
of the
Lys84(F7)
water
molecule, this
prosthetic
but is conserved in VHb.
Biochemical
properties
Hemoglobins
hemoglobins
generally
are
bacterial Hb and
and
flavohemoglobin proteins
intermolecular redox-domain in
(30) proposed
FHP.
an
electron
bridges
water
ring
FAD and
domain Glu384 is
highly
predominantly large
influence
may
jejuni
and
and
side
the
Lys84(F7)
C.
is
perfringens,
flavohemoglobins
are
binding
able to
properties,
hemoglobins
transferring
are
and Ser209, which side
heme and FAD groups.
flavohemoglobins
triade at the distal heme side in both
in the HMP
several residues
residues
These
associated with oxygen
oxygen. However, due to their structural
al.
binding
residues whose
domains.
respectively,
of
form
and Glu394 and the flavin
Thr and He in the Hb sequences of C.
replaced by
His85(F8)
Lys84(F7) forming a salt bridge. Lys84(F7)
polar
three
all
complex
Fe-02
in
hemoproteins (38).
In the NAD
moiety.
number of
from
of the heme iron
electron-transferring
an
HmpPa, Arg206, Tyr208
conserved and its side chain contacts
are
The
network.
flavohemoglobin proteins
flavohemoglobin proteins
of FAD, Gln207, which stabilizes
Gln384
properties
to constituents of
which contacts
Tyr209,
and
hemoglobin
domain of
chains contact the FMN
bond
hydrogen
structural differences around
interesting
and is present in all known bacterial
In the FAD
a
modulate the redox
can
oxygen storage and transport
enzyme
form
arrangement resembles the catalytic triad observed
the structural
cytochrome
may
and
and transport. Indeed
reversibly
i.e.
the
bind molecular
occurrence
and the presence of the
and
rather than
flavohemoglobins,
an
oxygen
of
a
catalytic
Ermler et
delivering
role for
Thus, this suggestion may question the role of bacterial hemoglobins and
flavohemoglobins
in
simple
oxygen storage and
25
delivery.
Introduction
Chapter 1:
Ligand binding
of O2, CO and NO
Bacterial
hemoglobins and flavohemoglobins
such
O2, CO, NO and CN
as
and the
,
oxidation state of the heme iron. The
and
for
the
with various
flavohemoglobins
binding
NO in
kinetics
contrary binds
are
are
The
reassociation kinetics after
of HMP
protein (106).
in the formation of
a
is
reversibility
ligand binding
given
in Table 3.
of
value for any other known
=
O2
to
a
confirm the
is
on
form in which heme and
oxygen reaction and
the
rapid
features
hemoglobin-like
protein
results
s-1),
(slow phase)).
More recent
laboratory (33).
changes
The
(44)
biphasic
in
very fast
higher
than the
data, however, indicate
(biphasic
are
and
a
(107) reported
which is several fold
The kinetic
et al.
hemoglobins
for VHb two different values
Orii and Webster
5600
hemoglobin.
reported by Giangiacomo
conformational
and
is not stable and
kinetics and affinities of
moderate rate of oxygen release from VHb
our
the
into
Interestingly,
(k0ff
in
reactivity
nitrosyl complex (61,106).
release of oxygen from VHb
VHb
oxy-complex
decays
photodissociation
published.
and 0.15 s_1
the
to
the oxidation state of the heme. E. coli
on
ferrous state. This
for oxygen release have been
phase)
Fe(III), although
and
Addition of dithionite and nitrite to the ferric
An overview of known
flavohemoglobins
only
form, and upon aerobic incubation with NAD(P)H, it
oxygenated
oxidized.
hemoglobins
of bacterial
Whereas oxygen and CO binds
exhaustion of NADH with residual oxygen
flavin
the
on
have been shown to be characteristic
ligands
Fe(II)
dependent
of those is
absorption spectra
strongly dependent
HMP is isolated in its ferric
reduced to the
to
ligands,
able to bind various heme
binding
hemoglobins (11, 61,106,144,149,155).
Fe(II),
are
release with koff
=
a more
4.2 s-1
(fast
parameters for oxygen release from
good agreement with
data obtained
behavior in oxygen release may be due to
with the presence
or
absence of
hydrogen bonding
of iron-
TyrBlO (44).
Replacement
properties
of distal GluE7
of VHb
by
(24). Binding
HisE7 has deleterious effects
on
of O2 to the mutated
is
became obvious from the absence of
distal His is
liganding
a
stable
protein
oxy-form,
thus
oxygen
binding
impaired,
suggesting
which
that the
the heme iron itself. However, the mutation had little effect
26
Chapter
on
effect
of VHb.
binding properties
CO
on
oxygen
Interestingly,
Binding
a
a
that the distal
Association
glutamine
does
hydrogen bond (24).
of oxygen and nitric oxide to various
Ligand pair")
of GluE7 to Leu had little
change
binding characteristics, indicating
not stabilize the bound oxygen with
Table 3.
1: Introduction
hemoglobin
and
Reference
[s_1]
Dissociation
[uM_1 s_1]
flavohemoglobin proteins.
o2
HMP
38
0.44
VHb
78
5000
4.2
200
(38)
(107)
(44)
(33)
(33)
(33)
(40)
(33)
(33)
(33)
(33)
(33)
(1.5)
117
VHb-Red
70
0.24
FHP
12
0.53
50
0.2
225
FHPg
Cj-Hb
Cp-Hb
HmpBs
HmpSt
132
1
197
2
39
7
9
2
HMP
26
0.0002
FHP
10-20
NO
Cj-Hb
Cp-Hb
HmpBs
HmpSt
Ligand binding to
the
HMP
can
bound
an
HMP has
such
0.002
41
0.0003
flavohemoglobins
as
oxygen
or
NO
external electron acceptor
a
NADH oxidase
NADH and to the
generation
On exhaustion of oxygen, the
Fe(II) heme, whereupon
acceptors
0.001
366
and
hemoglobins
mediate electron transfer either from the NADH via FAD to
ligand
FAD to
0.002
in its ferrous form.
protein
Enzymatic activities of
2
4
(38)
(40)
(33)
(33)
(33)
(33)
are
reduced
ferrisiderphores (2),
However, it
can
be
or
(115).
activity,
of
in the absence of
which leads to the
superoxide
ligand
in
and H2O2 in vitro and in vivo
vitro,
(113).
to
of
(96,100).
generate the deoxy
Various external electron
including dihydropteridine (148),
ferric citrate
(32), cytochrome
questioned,
if these reactions
27
from NADH via
rapid consumption
oxygenated species disappear
HMP
heme
In the presence of oxygen and NADH,
the flavin becomes reduced
by
a
a
c,
and
are
Fe(III)-hydroxamate (115).
of
biological significance
in
Chapter
dihydropteridine
vivo; the
negligible
since
more
1: Introduction
and ferric reductase activities have been
regarded
as
activities for these reactions exist in E. coli
potent enzymatic
(32,148).
Despite
of the lack of
reductase domain, VHb is also able to
a
reactions. Purified VHb is able to
NADH in
a
1:1
Nitric oxide
and
of
(39)
flavohemoglobins
identified in E. coli
an
inducible, O2 dependent, cyanide sensitive
protective enzymatic activity, which
aconitase.
of
Exposure
Gardner et al.
in the presence of
stoichiometry (151).
dioxygenase activity
Gardner et al.
generate hydrogen peroxide
perform catalytic
mechanism to HMP
which
expression,
(52)
shield
to
the
NO
sensitive
cells to NO elevated this
aerobically growing
and Hausladen et al.
(41)
able
is
activity.
attributed the observed detoxification
shown to
was
play
a
central role in the
inducible response to nitrosative stress. The authors stated that NO detoxification is
consuming O2
amounts to
and NADH and is
nitrate,
designating
the
NO and O2 in
converting
activity
nitric oxide
equistoichiometric
dioxygenase (NOD) (Figure
3).
The reaction kinetics and mechanism of HMP mediated NO
have been
(20°C)
and 240 s-1
of 1 uM
(20°C)
more
analyzed
(38, 40).
and 120 s-1
in details and the results showed
(37°C)
NOD
by
activity
of R.
eutropha
(37°C), respectively,
thus
temperature dependent than that of
activity approximately
NO when the ratio of
(100) reported
These two
that 35
findings
-
NOD
activity
of 94 s-1
at oxygen concentration of 200 uM and NO concentration
residue known to stabilize Fe2+-02
HMP
an
degrading activity
HMP
100 uM of O2
raise the
are
question
1:100
required
that NOD
Glu
activity
exceeding
determined to be 7.4 s4
is
or
of FHP is
Tyr29(B10),
a
His diminished NOD of
suggested
to be
(38). Furthermore,
hampered
Mills et al.
for maximal oxygenase
of whether HMP
28
activity
Substitution of
(40).
complex, by Phe,
is
was
showing
15-35 fold. NOD
NO/O2
FHP
provides
activity.
effective NO
1: Introduction
Chapter
removal under microaerobic conditions, since the natural environment of most
pathogenic
bacteria lacks
concentrations
which have
Figure
(41).
in the
are
micromolar to millimolar range
high
previously been proposed
(1)
(2)
(3)
(4)
NAD(P)H
overall:
2 NO
FAD
+
2Fe3+
+
FADH2
2Fe2+
+
2 02
2
Fe3+-02~
to
be
fe-
H+
+
-
concentrations,
highly inhibitory to HMP activity.
NAD(P)++ FADH2
—
2 Fe2++ FAD
+
2 H+
2Fe3+-02"
2Fe3+
2 NO
+
2 02
+
Furthermore, bacteriostatic NO
is low in oxygen.
or
+
2N03"
NAD(P)H
3. The reaction mechanism of HMP
Anaerobic conversion of NO and
+
catalyzed
NO detoxification
denitrosylase activity
2N03~
NAD(P)+
+
as
consumption
reduced to the
nitroxyl
dimeric intermediate
37°C),
of E. coli HMP
NAD(P)H.
anion
(NO ).
This
This reaction
(82).
(40).
efficiently
Latest results showed
then reacts
species
proceeds
at
novel
affinity
(51) reported
a
preferentially
The formation of
possibly
a
novel
a
biologically
turnover, the enzyme
heme-bound
O2),
can
which
was
a
uM NO,
determined
was
shown
identified to be
the
which is
approximately
29
that, due
inability
to bind
to the
a
and its
O2 in the ferric
reaction
(Fe(III))
subsequent
of the
at
(38).
state.
oxidation is
recently suggested
proceeds
a
higher
relevant O2 concentrations
Despite
denitrosylase
of HMP.
out
be found in the ferric
nitroxyl equivalent
function of HMP.
activity (38),
uM NO and 10 uM
activity
binds NO at
enzymatic
inhibition of HMP
activity
(1
third reaction of HMP with NO,
of ferrous HMP to NO relative to 02and the
During steady-state
NOD
rate of 0.14 s_1
N2O via
(36, 37).
Hausladen et al.
HMP
forming
enzymatic activity,
denitrosylase activity (O2 nitroxylase). They pointed
form,
a
NO under anoxic conditions and which
remove
rubredoxin reductase
Recently,
a
generation of
NO binds to the active site of HMP and is
which is several folds slower than the rate of the NOD
for HMP
to
of
Gardner et al.
by
outlined
Under anaerobic conditions the reaction of NO with HMP leads to the
nitrite under
H+
+
a
rate
NO
(at
40
50% slower than the rate of the
Introduction
Chapter 1:
Intracellular localization of VHb and HMP
Studies
the
on
the localization of VHb in
protein
exported
into
cytoplasmic
into the
explained by
and
the presence of
periplasmic
The
periplasm.
a
E. coli showed first
transgenic
during
export of VHb
16 amino acids
or
allocation of
space, and up to 30% of the
long
into the
protein
is
space is
periplasmic
sequence at the N-terminus of
into the
VHb, which may direct the protein partially
sequence is not cleaved
an
space. This
periplasmic
after the translocation process and is
intrinsic
an
part of the hemoglobin protein (75).
of the
Analysis
which
not
are
primary
overall
residue
of the central
lower than in
(60, 75).
signal
sequences. Instead of
residues at its amino terminus, it has
hydrophobicity
significantly
found in bacterial
typically
positively charged
typical
region
leader
VHb,
as
they
Furthermore, similar results
were
of this
peptides
Soluble fractions from
contain functional
putative signal
and
cytoplasm
allowed the export of alkaline
obtained in Vitreoscilla
phosphatase
coli.
The
allocation in both
overproducing
hmp using
that
30% of HMP
the
strain
Western
as
well
were
using
into the
in strain
periplasmic
and
periplasm
bearing only
a
are
amino
and
were
exclusively
E. coli
Recently,
In
phoA
(75).
space,
fraction of E.
found in
a
HMP
chromosomal copy of
of both fractions revealed
found
in
the
cytoplasmic
highly
similar to
(148).
Webster
and
immunogold labeling
The results
to
cytoplasmic
fraction. The N-terminal sequence of HMP has been shown to be
that of VHb
blotting.
acids)
periplasmic
single
blotting. However, spectral analysis
CO-binding hemoproteins
charged
found to
were
Western
periplasm of
recovered from the
cytoplasm
as
a
able to abduct carbon monoxide in vitro.
were
E. coli HMP has been found to also allocate into
approximately
The
sequence is
due to the presence of
periplasm
having
negative charge.
a
addition, fusions of the N-terminal sequence of VHb (16-23
and
features,
structure of this sequence reveals several unusual
clearly
coworkers
(121)
analyzed
VHb
localization
both in Vitreoscilla and recombinant E. coli
indicate that VHb has
mainly
30
a
cytoplasmic
expressing
and not
using
VHb.
periplasmic
Introduction
Chapter 1:
localization in both
suggest that the observed VHb
The authors
organisms (121).
export by Khosla and Bailey (75) may be due
to the
VHb. In addition, it cannot be ruled out that the osmotic shock
the
periplasmic sample preparation generated
Regulation
and function of bacterial
and extrusion of
overexpression
these
procedure
used for
false results.
putatively
and
flavohemoglobins
hemoglobins
Vitreoscilla
The
hemoglobin
VHb
oxygen-dependent promoter
limited conditions in Vitreoscilla
of vhb
functional
in
Azotobacter and Rhizobium
Rhizobium
etli
(122)
(23), Streptomyces
Burkholderia
saturation in both Vitreoscilla and E. coli
activated
positively
by
FNR
modulated
sp.
(92),
and
by
CRP.
In
(74, 76). Activity
the
coli FNR
from Pvhb
strains that
catabolite
expression
such
as
unable to
were
synthesize
repression, glucose
those encountered in
conditions
molecules
(141).
are
expression
The DNA
highly
switching
promoters
CRP
e.
induced
under
less than 2%
of air
of Pvhb in E. coli has been
expression from Pvhb
or
consensus
cAMP
was
DNA
a
was
motif is
sequences of either £.
substantially
(74). Despite
glycerol-containing
and
is
(156, 157),
reduced in
the influence of
media gave
comparable
levels of reporter enzymes from Pvhb under carbon-limited conditions
E. coli strain the
that
(74,141). Expression
marcescens
promoter sequence
to the
E. coli CRP
completely
(74), Pseudomonas,
maximally
Pvhb is
encountered, which reveals high similarities
or
Serratia
CRP and FNR, i.
by
of the vhb gene. The
coli
E.
as
conditions, when dissolved oxygen level
microaerobic
shown to be
such
(108).
sp.,
expression
and has also been shown to be
(74),
heterologous hosts,
various
which is induced under oxygen-
controls the
(153),
Pvhb has been characterized in E. coli
(Pvhb),
can
typical
fed-batch fermentations
from Pvhb
binding
was
of the
only
in 1 base
noncongruent base pair
result in their in vivo
global
pairing.
an
fnr negative
the
E. coli
regulatory
It has been shown
in the half-site
regulation by
31
In
reduced 2-fold under microaerobic
sequences of these two
identical and differ
(78).
motifs of
target
corresponding noncognate
Chapter
Thus it would
regulator (130).
1: Introduction
possible
seem
that E. coli FNR and CRP
bind to the identical site in Pvhb- The FNR
structure
around
latter
has been
divergently allocated,
position -41.5,
binding
site is not
non-E. coli FNR
addition of
Joshi
whereas
sharing
consensus
complex nitrogen
a
resulting in a third level
of
Tsai et al.
such
homology
reporter protein accounted up
well
the manifold
as
for
interesting
induction is
bioreactor. Thus,
proposed by
diffusion and
Based
that
the
chemical inducers
be omitted
can
also resulted in enhanced
Khosla et al.
(78)
cytochrome
o
will
complex (aerobic
ratio, and is able
relative to
of
of
VHb
processes since the
(78).
terminal
terminal
that VHb
oxidases
oxidase)
generate
cytochrome
higher
final cell
was
not restricted to
expression could
(76).
growth
protein synthesis (78). Therefore,
increases
predominantly
to
extremely
it has
facilitate oxygen
aerobic metabolism in E. coli.
expression
activity
the medium,
controls under oxygen limited conditions
(70)
formulated
d
a
and
a
hypothesis stating
intracellular
the
concentration under microaerobic conditions. This increase
relative
the
dissolved oxygen concentrations in the
mathematical model, Kallio et al.
on a
by
protein. High level expression
showed that the effect of VHb
improve
to
mechanisms have rendered Pvhb
by lowering
plasmid bearing
experiments
improvements but
been
extract
of VHb in E. coli, recombinant cells grew to
densities relative to
Further
to the known E. coli and
biotechnological production
in
achieved
expensive
Upon expression
site
induction, the level of expressed
to 10% of total cellular
regulatory
applications
readily
binding
regulation of Pvhb (78).
In bioreactor cultivations under conditions of full
as
promoter
of Pvhb is achieved
yeast
as
located this
able to
located it around -23.5. The
(129). Repression
source
site within the
(141)
(68)
and Dikshit
any sequence
sequences
binding
are
the
effective
thereby
energetically
has
higher proton
translocation
larger proton gradient
complexes (microaerobic
32
across
terminal
would shift the
more
be used in microaerobic E. coli.
oxygen
favorable
Cytochrome
o
activity, H/0+
the cell membrane
oxidase) (119,120).
1: Introduction
Chapter
This
hypothesis
either
has been studied in details and
cytochrome
cytochrome
o
and
a
o
cytochrome
or
1.5-fold increase in
coli relative to
hemoglobin-free
to
50%
generate
a
VHb-negative
a
65%
lacking
higher
flux
ATP turnover rate, thus
Dikshit et al.
could
oxidases
these results
the
steady
tension the
through
using
strains relative to control cells
more
through
slowly. Therefore,
expression
the
indeed able
are
a
30%
a
higher
aerobic
the
a
ATP
the model of
showed that
on
E.
an
E. coli
substrate
to express VHb. These results
a
not been
state level of cellular
known to reduce electron flow
consumed
VHb-positive
terminal
oxidase
investigated
function.
further and
we
lacking
the
different E. coli strain
(Kallio P., personal communication).
terminal oxidases
expressing
in
supporting
(26)
grow
engineered
were
However, this potential function of VHb has
Additionally,
5-fold increase in
cells
leads to
thereby
suggest therefore that VHb itself may contain
verify
complexes
lacking
flux per reduced oxygen molecule relative to
succinate and lactate when the cells
have not been able to
a
(142). VHb-expressing
higher proton
both terminal
d
cytochrome
(14, 70, 142). Furthermore,
Kallio et al.
mutant
and
of VHb in cells
revealed
complexes
controls
higher proton
control. The
synthase activity
d
expression
of the oxygen
respiratory
chain
in
one
NAD(P)H
near
the
anoxic conditions
respiratory chain,
may
hypothesize
delivering
E.
is 1.8-fold lower in VHb-
so
(143).
that
Anoxia is
NAD(P)H
is
that under low oxygen
VHb may increase the electron flux
coli, thus reducing the level of cellular
NAD(P)H.
Furthermore, VHb-positive cells display increased
active 70S ribosome
also
complexes
accompanied by
a
amounts of both tRNA's and
under microaerobic conditions in E. coli. This
corresponding
increase of
a
marker enzyme
was
activity (103).
Thus, the upregulation of VHb expression and the observed positive effects of VHb
expression
in cells grown under oxygen limited conditions
VHb is able to stimulate the energy
efficiency
of VHb
production
expressing cells.
33
and
thereby
clearly
indicate that
increase the metabolic
1: Introduction
Chapter
R.
eutropha
FHP
FHP
is 20-fold
expression
eutropha (118).
Two
noncoding region
two
upregulated
molecules suggests
global regulatory
eutropha. However, isogenic flip
of R.
aerobic
or
did not accumulate nitrous oxide
acceptor.
Altogether,
NOD
these results
gas metabolism
et al.
the
Recently,
flavohemoglobin
significant
differences in
type, however, the
has
FHP
eutropha
or
indirectly
proposed
earlier
Poole et al.
(Ill)
as
electron
as
demonstrated
been
directly
FHP interacts
mutant
(40).
with the
by
Cramm
HMP
roles of E. coli HMP
To elucidate the
possible
response of the
hmp promoter (Phmp)
found to be
responsive
mutant and
region (93).
In
a
to iron
depletion.
putative FNR-binding
site
conditions.
was
is enhanced in
expression
at
Phmp
positions
-2 to +11 in
an
Phmp
inducer under aerobic conditions. The
were
of these
nitrogen compounds
regulatory proteins (111). Furthermore,
expression
was
of RNA
The
upregulation
by
the
of NarLP and SoxRS
independent
growth
a
observed when cells
This induction is mediated
(cts)
to be
occurs
studied the
addition, HMP expression is stimulated directly by the presence of
effects
phase.
growth
Anaerobic
an
HMP
protein
under various
nitrate and nitrite; nitric oxide is also
were
state
dependent
entering
the
induction of
stationary growth
stationary phase-specific sigma
subunit
polymerase (93).
dependent
of
hmp expression by
on
biosynthetic pathway
the
these
(18).
E. coli
fnr
of
denitrification in R.
during
no
denitrification with nitrite
during
suggest that
show
with the wild
activity
by
role of FHP in the anaerobic metabolism
a
mutants
growth. Compared
anaerobic
upstream
of FHP
regulation
have been identified and the
flip
conditions in R.
oxygen-limited
motifs for NarL and FNR in the
potential binding
of
under
NO
was
the MetR, which is the
in E. coli.
Interestingly,
intergenic region between hmp
and
glyA,
34
studied in
more
global regulator
two MetR
which
are
binding
details and shown
of the methionine
sites
divergently
are
located in
transcribed
(95).
Chapter 1:
Homocysteine
(mefL),
is
a
co-regulator
Introduction
growing
cultures resistance
NO-compounds. Upon feeding
against
nitrosative stress
been shown to be necessary for induction of
occurs
depletion
upon
region.
Inactivation of
homocysteine may
Membrillo-Hernândez et al.
in the presence of
as
(97)
also
the
shown
reported
a
using
coli
E.
independence
of
mutants
these
even a
28-fold induction of
in the presence of
paraquat,
an
hmp-lacZ
treatment
a
MetR has
(21).
SNP and GSNO but
induction
induced
of HMP
(95).
a
on
fusion after
that induced HMP
fusion
subtler pattern,
only by paraquat
SoxRS
upregulation by
transcriptional
two
promoter
hmp-lacZ
an
a
activators.
(97). Anjum
as
only
Induction
(95).
nitrosation reaction
Furthermore, hmp regulation by paraquat is dependent
showed
restored
MetR to bind to the
was
to the
homocysteine
the induction of
of which
expression
concentrations above 200 mM. The
was
via
occur
of
paraquat. The induction of sodA-lacZ followed
hmp-lacZ,
did the
hmp
homocysteine, which allows
of
was
hmp-lacZ by
site is essential for
binding
the downstream MetR
made these
homocysteine synthesis,
the function of which is also needed for
mutants sensitive to various
pathway
for MetR and mutations in the methionine
prolonged
expression
et al.
(3)
incubation
spectrally
to
detectable levels.
The observation that HMP is
superoxide radicals
a
NADH oxidase and is
and H2O2 led to the
hypothesis that HMP might be
Interestingly,
the oxidative stress response in E. coli.
able to
thereby
HMP is not
generate
involved in
only generating
ROS, but is also needed for full expression of SodA and SoxS. Furthermore,
mutant
was
markedly
more
paraquat concentrations,
Hughes (112) suggested
leading to
the full
to
degrade
which
against
shown
that HMP
expression
Several lines of evidence
and resistance
as
towards
sensitive
by
a
viability
might
act
paraquat, particularly
assay
as an
(94) Therefore,
amplifier
of
hmp
a
lower
at
Poole and
superoxide
stress
of SoxRS stress response.
clearly
prove the
implications
of HMP in the response to
nitrosative stress. First, it is well established that HMP is able
NO under aerobic conditions,
catalytic activity, dioxygenase
although
or
35
it has to be
denitrosylase,
is
analyzed
more
in
details,
relevant in vivo.
Chapter
1: Introduction
Second, in vivo experiments revealed the important role of
against
that
various
agents producing
cells
hmp
compounds. Third,
Various classes of
Fe-S
The
expression of HMP
proteins
are
is induced
considered to be
protective activity
(39),
revealed very
These
proteins
effect of HMP
of HMP
was
whereas the
extremely
of heme and Cu
both terminal oxidases
(cytochrome
o
sensitive to NO. However,
NO inhibition.
(132) reported
and
d)
of
containing
containing proteins
targets of
Additionally, respiration
an
in
a
protective
mutant strain
a
hmp
mutant
was
had to admit that due to the reduced
they
of HMP under microaerobic conditions
activity
main
are
as
Cu/Fe proteins.
and various
contain both heme and Cu. Stevanin et al.
on
nitrosative stress, such
demonstrated for the Fe-S
protection
these
NO in E. coli.
by
targets of
Terminal oxidases
recently.
either terminal oxidase.
lacking
already
provided by
thread
the
towards
containing proteins, heme-containing proteins,
enzyme aconitase
was
the
experiments showed
nitrosative stress. Various
sensitive
more
are
HMP in the resistance
an
enhanced NO'
toxicity
is to be
expected.
Alternatively,
E.
coli,
Poole et al.
since the
limiting
binding
that HMP could act
(114) proposed
causes
steady
a
mechanism
ability
might
activation of the anaerobic gene
state.
oxygenated
dissociation of the
flavin reduction. Due to the
fashion, such
oxygen
sensor
in
of oxygen modulates the reduction level of FAD, thus
flavin reduction in the aerobic
concentration
as an
HMP
of HMP to reduce
control
species
Fe(III)
Fe(III) reduction,
regulator FNR in E.
of the oxygen
Lowering
in
a
and sustained
flavin mediated
which is
required
for
coli.
Flavohemoglobins from various bacterial organisms
The
expression
conditions such
of
as
oxygen
regulatory proteins
expression
many
hemoproteins
deprivation
have been
in various
or
is
as
the
36
by
environmental
stress
oxidative and nitrosative stress. Several
implicated
bacteria, such
induced
in the control
global
of
hemoprotein
anaerobic response
gene
regulator
Chapter
1: Introduction
phase responsive
FNR, RedD/E, Fur, NAR, MetR and stationary
levels
Interestingly,
general,
in
mediated
independently
positively
either
are
induction of
modulated
negatively
or
ROS and RNS
hmp expression by
of the oxidative stress
by
(19, 57, 85).
is increased in the presence of RNS and
Furthermore, expression of these genes
expression
as
ROS.
by
mainly
was
SoxRS and
responsive
OxyR
transcription factors (19, 57,85).
Early support
component of
hypothesis
to the
a
stress response
hmpX
upon inactivation of
became unable to infect
reduction
determinants of E.
of PL
was
hmpX
of
upregulation
of
presence
a
tobacco
(35).
fusion
growing body
flavohemoglobins
be
of
great
for
clinically
typhimurium
take the
host.
Bearing
resistant
showed that
(35). They
chrysanthemi,
which
(PL),
the bacteria
pathogenicity
are
factors
host
plant
on
cocultivation of E.
by
culture.
The
infecting plants.
as
synthesis
regulation
shown
chrysanthemi
by
transcription
of
-compounds
in the
likely
that
of
the
in the
conditions per
Microaerobic
and therefore it is very
This
interest
and S.
potential
integral
This behavior could be attributed to the
not sufficient to start
against
general.
of
a
an
se,
as
hmpX-gus
plant
hmpX expression
those radicals.
a
in the response to the thread with NO radicals and
flavohemoproteins
of
as
of evidence has accumulated in the last few years that showed
nitrosative stress in
might
were
(10);
contributes to the resistance
role of
E.
In the meantime the involvement of NO"
defense has been shown
A
fusion
suspension
encountered upon infection,
et al.
plant pathogen
mutant when
dependent
be
function
in conditions of low oxygen tension. The
hmpX
hmpX-gus
an
reported by Favey
pectate lyases
of
chrysanthemi
shown to
flavohemoproteins
plant species.
abolished in the
was
was
gene in the
synthesis
the
in
that
general
the
clinical
relevant
advantage
role of
flavohemoprotein
microbiology.
microorganisms,
such
As
as
in NO defense
shown
above
M. tuberculosis
of this resistance mechanism upon infection
in mind the
steadily increasing
pathogens, flavohemoproteins would
37
offer
a
novel
number of antibiotics
drug target for therapy.
Chapter
Hemoglobins
The commercial
and
production
the
and mammalian cells.
especially during large-scale
demand for oxygen
bioreactor
of
overexpression capacity
can
be
configurations,
has very low
Oxygen
and
of
important
oxygen-requiring bacteria,
solubility
in water and various
types have high nutritional demand for oxygen
cell
high
density production
satisfied
partially
e.g.
biotechnology
of desirable metabolites and
variety
a
and cultured cell
microorganisms
in
flavohemoglobins
pharmaceuticals employ
fungi
1: Introduction
by improving
improved mixing
rates,
high
processes. The
process
parameters and
high-efficiency dispersion
systems and modifications of the medium (83).
Use of VHb to
The metabolic
effects
improve
engineering approach
based
was
Vitreoscilla expresses
The
limited
effects of
growth
documented
extended
in
was
(Table 4).
The
collaboration
use
of VHb
with
a
highly
mycelial pellets
the
viscous
media,
was
on
(12).
VHb
production resulting
in
relative to the
strain
original
an
The
plants.
deprivation
transferred to E. coli
successfully
promoting
in
the
and
numerous
efficiency
organisms
expression technology
biotechnological industry
to
strain. This strain has been
since the
it has been
will be limited. The vhb gene
chromosome
and
protein
enhanced under microaerobic conditions
erythromycin production. However,
growing
to alleviate adverse
hemoglobin VHb, growth
VHb-expression
significant Saccharopolyspora erythraea
for
genetic strategies
protein production
and
productivity
observation that under oxygen
bacterial
of recombinant E. coli
positive
seeks
This gene
hemoglobin.
of the
and
microorganisms, higher cells,
previous
on
expression
upon
production
79).
growth
of oxygen limitation in
approach
and
cell
was
therefore
space-time yield
(101).
38
well
are
successfully
an
industrially
optimized
before
bacterium is
that oxygen transfer to the
expression significantly
enhanced
of oxygen-
has
mycelium-forming
argued
(76,
stably integrated
increased
of
into
erythromycin
approximately
100%
Chapter
1: Introduction
Furthermore, the beneficial effects of VHb expression technology
to be restricted to bacterial
of natural
production
to
of metabolites
(58). Therefore,
of
plants
has
compounds
Farrés and
cultured tobacco cells. VHb
suspension
use
cells is known to affect both cell
Oxygen supply
phase
The
recombinant
or
plant
organisms.
Kallio
plants
gained
have
shown not
cells for the
momentum
growth and
(34)
expression
and
were
the
production
VHb in
expressed
resulted in
a
(28).
shortened
lag-
and increased final cell densities in batch cultures relative to control cultures.
Table 4.
Summary
of beneficial effects of VHb
in various
organisms
References
Effects
Organism
Acremonium
expression
3.2-fold increase of
chrysogenum
cephalosporin
30% increase of neutral
Bacillus subtilis
amylase activity,
protease,
C
(22)
production
7-15% increase of
and 1.5-fold increase of
a-
protein secretion
15% increase of biomass, and 2-fold strain DNT increase in
Burkholderia sp
degradation
40%-100% increase of tissue plasminogen activator
production
30% increase of L-lysine titer, and 24% increase of L-lysine
the rate of DNT
Chinese hamster ovary
cells
Corynebactenum
glutamicum
yield
Escherichia coll
protein
protein
30% increase of total cell protein
80% increase of CAT activity
40% increase of ß-galactosidase activity
61 % increase of ß-lactamase activity
Escherichia coli
3.3-fold increase of
Escherichia coli
1.8-fold increase in ferritin
Nicohana tabaccum
more
2.2-fold increase of total cell
Escherichia coli
Escherichia coli
Escherichia colt
Escherichia coli
50% faster
a-amylase activity
production
germination
dry weight,
growth 80-100%
chlorophyll, 34% more
rate, enhanced
30-40%
more
(102,108)
(109)
(124)
(76)
(141)
(79)
(79)
(79)
(103)
(29, 80, 88)
(16)
2.1-fold increase of total cell
Escherichia coli
(69)
,
(56)
nicotine
Nicotiana tabaccum,
Enhanced
suspension culture
11%
Pseudomonas aeruginosa
mcrease
shortened
Saccharomyces cerevisiae
Saccharopolyspora erythraea
of viable cell number, increased oxygen
Enhancement of acetoin, and
marcescens
Streptomyces
Streptomyces
Streptomyces
Xantomonas
of bean
plants,
2,3-butanediol production,
10-fold increase of actinorhodin
production
density
2.2-fold increase of Oxytetracycline production
15% increase of viable cell number, enhanced degradation
coehcolor
50% increase of final cell
nmosus
maltophila
of benzoic acid
The above-cited results show that
a
heterologous
wide range of distinct
39
VHb
(42,89)
(122)
(14)
(12,101)
increase in cell size
hvidans
oxygen limitation in
(34)
lag phase
uptake
68% higher nitrogenase activity in nodules
and 53% higher total nitrogen content
3-fold increase of final cell density
70% increase of final erythromycin titer
Rhizobium eth
Serratia
growth,
expression
organisms. Thus,
(156,157)
(92)
(92)
(5)
(90)
is able to
cure
the inverse metabolic
Chapter
engineering approach,
i.
organisms, represents
limitation
Novel
a
the
genetic
fast
means
for
hemoglobin proteins
It has been
hypothesized
that the
generate
have been
a
number of
alleviate the adverse effects of oxygen
to
improved performance
analyzed
performed
Andersson
in
not
are
optimized
were
to elucidate the
physiological
Glul37(H23)
Gln66(E20)
50%
(Ala56(E10)
with
extension of
eight
indicate that the
pVM50
are
cloning strategy
residues
positive
sequence reduced cell
Bollinger
et
al.
(8)
effects elicited
expressing
growth
Gly),
to
of E. coli
155%
and
Gly),
to
by
mutant
peptide
growth approximately
were
screened for
The
proteins
and
by approximately
22%
Arg36
and
(His36(Cl)
90%
to
(Ile24(B5)
to
Thr),
proteins
also contained
experiments
from
VHb-expression
an
also
pVM20
tail. Removal of the N-terminal
23% and 53%,
respectively,
relative to
(1).
screened
various
microbial
hemoglobins
biotechnological production
oxygen-limited
to
at their N-termini. The
(MTMITPSF)
uncharacterized and unidentified
be used in
PCR
error-prone
effects of novel VHb
the mutated VHb
linked to the N-terminal
native VHb controls
for
concomitant decrease of acetate excretion into the culture
a
medium. Due to the
used
mutants with
by pVM20, pVM50, pVM104,
carried
and
respectively,
(1)
foreign
VHb-technology
vhb genes. VHb mutants
(mutations: Glul9(A17)
Arg66),
al.
et
able to enhance microaerobic
to
to ameliorate the
for
under microaerobic conditions and several clones
of four VHb mutants,
expression
cells
of VHb
properties
randomly mutated
improved growth properties
pVM134,
heterologous
to
cells, screening of novel globin genes and generation of VHb
biotechnological applications.
and
phenotypes
hosts
improved properties
were
transfer of useful
(5).
heterologous
hosts
e.
1: Introduction
and
hemoproteins
were
40
for
the
existence
flavohemoglobins,
processes and
conditions. Growth behavior and
various
genomes
to
enhance cell
byproduct
analyzed
of
which could
growth
under
formation of E. coli
in microaerobic fed-batch
Chapter
1: Introduction
expressing flavohemoglobins
cultivations. E. coli cells
and D. radiodurans grew to similar final cell densities,
VHb.
K.
the clones
Although,
pneumoniae
the
or
from P.
as
expressing flavohemoglobins
hemoglobin
from C.
S.
aeruginosa,
did the strain
typhi,
expressing
from E. coli, B. subtilis, and
the
jejuni, outperformed
plasmid bearing
control strain, these clones did not reach the final cell densities measured for VHb.
Additionally,
yield
increased
recombinant strains, and
with D. radiodurans
carrying
an
approximately
glucose
on
2-fold
flavohemoglobin-expressing
hemoprotein expressing
The
improvements
engineered
biomass
yield
expression
to express
as
are
that
obtained
recombinant
these
myoglobin
growth
coli
strain
was
control strain
more
did not result in any
growth. Interestingly,
plasmid bearing
all
E. coli relative to the E. coli control
yeast flavohemoglobin (Yfb)
the
was
for
able to utilize the carbon substrate
of horse heart
microaerobic
during
final cell densities
strains
measured
was
enhancement
parental plasmid only. Thus, showing
the
efficiently (8).
of
an
E.
not able to reach similar
(71).
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Intracellular expression of
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Expression
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Hemoglobin-Reductase
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50
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Chapter
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terminal)
domain with
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E.
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Approximately
the
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final cell
same
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active
site
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or
and
expression
cassettes
were
hemoglobin (FHPg), FHPg-FAD, FHPg-
active
hemeproteins
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their
were
ability
produced
from all
abduct carbon
to
of VHb-FAD-NAD-reductase increased the final
E. coli cells
densities
relative to the
approximately
expression
of VHb-NAD
approximately
recombinant
control.
51
synthesizing VHb.
or
or
FHPg-FAD
expressing
increased the
control under the
FHPg-NAD
20% relative to the
ß-lactamase
50% and 75%,
achieved with the E. coli strains
were
VHb-expressing
VHb-FAD-NAD-reductase
more
redox
reductase
encoding NAD(P)H, FAD,
and VHb. The presence of VHb-FAD
2.3-fold
(FAD)
(carboxy-
in frame after the vhb gene.
eutrophus
indicated
optical
reduced the final cell densities
second
effects which VHb exerts in
positive
modified
was
as
the
fused sequences
wild-type
final
density slightly
The
a
fed-batch culture relative to the control
cultivation conditions. The
control.
we
Biochemically
monoxide. The presence of FHP
density
improve
the amino terminal
FHP activities.
these constructions
cell
carboxy-terminal
the
reductase activities of A.
gene
with VHb. However,
and flavin adenine dinucleotide
be used to
microaerobic Escherichia coli cells,
NAD(P)H-FAD
terminal-hemoglobin
and unlike VHb, FHP possesses
whether
examine
homology
has 51% sequence
NAD(P)H
and
growth
gram-negative hydrogen-oxidizing
of the
flavohemoprotein (FHP)
bacterium
microaerobic cell
improve
formation. The amino
oxygen-dependent product
domain of the
from Vitreoscilla sp. in
strain
proteins
VHb-expressing
was
relative to the
fusion
same
also
able
to
VHb-expressing
Chapter
2:
Heterologous hemoglobin expression
to alleviate
hypoxic
cell
growth
Introduction
One of the foremost
of useful
phenotypes
and
the exact mechanism
has been
hypothesized that,
from solution and
(18, 45, 49).
articles
productivity (See
and release
(Kd
=
72
provide
by
uM) (45),
and
having
did
some
a
family
pyridine
in
horse heart
coli in
and
and
were
be
can
myoglobin
cytoplasmic flavohemoglobin
has
A.
eutrophus
respiratory type
in central
using engineered
to enhance
applied
specific
hypoxic
either of two different
flavohemoglobin,
each
globins containing
(51).
One such
was
such
as
protein,
a
coli
(43),
Erwinia
Fusarium oxysporum
megaplasmid
also identified from
bacteria
binding
have been identified
E.
norvegensis (15),
Candida
(FHP),
N-terminal oxygen
flavohemoproteins
(23),
(19).
a
encoded
facultatively
Alcaligenes eutrophus
grows, like Vitreoscilla, in oxygen-scarce environments and
of metabolism, but A.
oxygen, if either nitrate
Limited oxygen
and
changes
conducted
and yeast
lithoautotrophic, hydrogen oxidizing, gram-negative
(4, 30, 48).
expression
expressing
eukaryotic organisms
cerevisiae
o
fed-batch cultivations
oxygen-limited
Bacillus subtilis
Saccharomyces
physiological changes
with VHb, grew to lower final cell densities than
of two-domain
prokaryotic
chrysanthemi (12),
(37)
heterologous organisms
nucleotides and
levels. E. coli cells
and C-terminal reductase activities, termed
both
cytochrome
besides VHb
globins
homology
sequence
VHb-expressing E.
Recently,
parameters for oxygen
in Escherichia coli indicate increased overall ATP
(7,18, 27, 38-40). Experiments
hemoglobin-like proteins,
to enhance their
these effects is unknown, it
them for cellular activities in
protein production
Vitreoscilla
VHb is able to scavenge oxygen molecules
and turnover rates, increase in
E. coli to determine if
growth
causes
More detailed observations of biochemical and
carbon metabolism
of
transfer
2, 6, 14, 25 summarizing the results).
which VHb
decreased levels of reduced
activity,
expression
due to its unusual kinetic
accompanying VHb-expression
production
is
bacteria, yeast, fungi and plants
Although
binding
engineering (the genetic
heterologous organisms)
to
in aerobic
hemoglobin (VHb)
growth
of inverse metabolic
examples
supply
or
nitrite
causes
are
eutrophus
present
as
approximately
52
is also able to grow without
terminal electron acceptors
(4, 30).
20-fold increase in FHP content,
Chapter
that
suggesting
regulated by
oxygen
high
modules:
of 403 amino acids
266
(Ferredoxin
NADP+
with
identical
(Fumarate
binding
high
connected
and
by
Reduction),
which controls the
(42). proteins.
complex
of the
well
goal
-
258)
homology
to
C-terminal
and for NAD+
FNR-like
the
proteins
not
are
regulator
FNR
for
The reductase domain is able to
as
cytochrome
capable
of
c.
Furthermore, the
reversibly binding
The different domains of the FHP
short sequences of 5-7 amino acids
between
domains, respectively (8). The three-dimensional
The
153
a
protein
expression of genes required
domain is
FHPg
(30, 31).
as
monomeric
a
and
147),
transcriptional
global
Phe259-His-Ile-Asp-Val-Asp-Ala265
resolved
-
(residues
sequence
publication)
of two different
well-characterized
oxygen in its reduced state
are
FHP is
The FNR-like
reduce several artificial electron acceptors
heme-iron
residues 1
sites for FAD
share
anaerobic metabolism in E. coli
fr-type
Vitreoscilla is
in this
Reductase) protein family.
the
Nitrate
(8, 44).
(44.8 kDa) consisting
hemoglobin- (FHPg;
which
403),
-
growth
like VHb in
(termed FHPg
with VHb
homology (51%)
redox active domain with
(residues
domain of FHP
hemoglobin
N-terminal
a
cell
(3, 31).
sequence
polypeptide
hypoxic
alleviate
eutrophus,
of FHP in A.
expression
The N-terminal
shares
Heterologous hemoglobin expression to
2:
polypeptide
Prol48-Gly-Gly-Trp-Lysl52
FHPg-FAD
and
FAD-NAD
structure of the FHP has also been
by X-ray crystallography (10).
of this research
expression
of
FHPg,
was
to
study potential biotechnological applications
FHP and fusion
parts thereof (NAD+/FAD domains)
of the
in E. coli
bioreactor. In addition, the vhb gene
under microaerobic culture conditions in
a
fused with sequences
FAD and FAD-NAD activities of the C-
encoding NAD+,
terminal domain of FHP.
these different
VHb-
Hypoxic growth
heterologous
expressing
E.
and fusion
of different E. coli constructs
proteins
was
compared
with
coli, reaching higher final cell densities relative
carrying VHb-negative
E. coli
(21),
in
a
controlled
53
hypoxic
bioreactor.
was
expressing
growth
to
of
plasmid
Chapter
2:
alleviate
Heterologous hemoglobin expression to
hypoxic
cell
growth
Materials and Methods
E. coli strains and
E.
plasmids.
[F~
coli DH5a
(rk~ mk+) supE44
hsdR17
endAl
Gibco BRL Life
(<|)80dZacZAM15) (lacZYA-argF) U169;
host
the
during
Laboratory]
was
subcloning steps.
reductase constructions
for
used
expression
was
by
pPPCl,
a
a
Cold
[AT, F";
gift
pKQV4
(19). pGE276 (8)
used
was
Spring
as a
Harbor
microaerobic bioreactor.
pRED2
of other
and
globin
(21, 22). pUC19 (50)
an
as
containing
IPTG-inducible
the vhb gene, has
contains the flip gene of A.
from Dr. B. Friedrich
relAl
globin-
or
pKQV4 (36) employed
derivative of
Kallio et al.
generous
a
and
subcloning
vector.
been described
and
of cells in
Technologies]
gryA96
and
the effect of VHb
growth
on
coli MG1655
E.
X~ recAl
the Vitreoscilla vhb gene has been described elsewhere
carrying
was
analyze
used to
thi-1
eutrophus
of Berlin,
(Humboldt-University
Berlin, Germany).
DNA
manipulations.
All restriction endonucleases
according
according
PCR
recommended
to the
to standard
technique (32)
accession
reading
no.
9600 PCR
was
coding
combinations. PCR
System
for PCR reactions
purchased
protocols.
for
or
structures
oligonucleotides
suppliers
manipulations
DNA
FHPg, NAD,
and Pwo
and used
performed
were
were
encoding
accession
and FAD
no.
protein
performed using
a
VHb
(GeneBank
X74334) (8)
and open
domains of FHP
their
Amp
Polymerase (Boehringer Mannheim). Oligonucleotides
new
designed
various domains of fusion
or
Perkin Elmer Gene
synthesized by Microsynth (Balgach, Switzerland)
encoding
were
the genes
(GeneBank
FHP
amplifications
were
amplify
used to
(CT A,
TT A)
proteins
if
shown in Table 1. Translational stop codons
gene
from commercial
protocols (33).
X13516) (22)
frames
were
fusion
in
a
way that the
54
(Table 1).
The
sequences between the
remained
original
are
also inserted into the
necessary
hinge
proteins (globin-reductase)
and that amino acid sequences relative to the
were
and
highly
conserved
sequence of FHP
were
Chapter
minimally
FHPg
or
2:
Heterologous hemoglobin expression
altered
protein
FAD-NAD fusion
proteins.
Leu to Phe
fragments
were
Switzerland).
recovered
Table 1.
Figure 1).
(F)
This
DNA
fragments
Oligonucleotides
stop
of the
vhb,fhp,
and the open
NAD and FAD subunits
reading frames
à)
5: GGGGTACCGGGTTCAACCGCTTGAGCGTAC
vhb
6: GGGGTACCTGGAAGGGGrGGCGCACCriTGTG
fad
7: ACGTTGACGCCAAGACACCCATTGTnCCTGATC
nad
8: GCGTCAACGTCGATATGGAATTCAACCGC7TGAGCGTAC
vhb
9: CGTCAACGTCGATATGGGGTTGTTCCGCAGAGCGCTC
flip
lOTGCACTGCAGTGCACTATTAGCrrCCATAGGGCGCAG
Restriction sites, translational start
and the
and
vhb
start
Pstl
a)
gel electrophoresis
4: CGGAATTCCGATGTTAGACCAGCAAA CCA 7T
HmcII
Oligo
(Qiagen, Basel,
nad
2x
Hindi
Oligo
gene
3: TGCACTGCAGTGCATTACTACTCAGCAAACAGGTCG
Hindi
Oligo
agarose
amplified
flip
2x
stop
Kpnl
Oligo
PCR
FHPg domains
flip
Kpnl
Oligo
B).
KpnI.
2: TGCACTGCAGTGCACTATTATTGTTCCGCAGAGCGCTC
EcoRI
Oligo
1
or
PCR Purification Kit
amplifications
start
Pstl
Oligo
either with VHb
separated by
encoding FHPg,
Pstl
Oligo
restriction site for
1: CGGAATTCCGATGCTGA CCCAGAAAACAAAAG
EcoRI
Oligo
a new
Extraction Kit.
used for PCR
of flip
respectively. Thus, Glyl50
(P), respectively (Figure
were
growth
to Thrl49 in VHb-FAD and VHb-
change generates
purified using QIAquick
cell
The amino acid sequence of either
changed
(LHIDVDA)
and Pro
using QIAquick Gel
Oligo
was
sequence
of NAD domain
linkage
changed
in
VHb domain contains 147 and 146 residues,
within the FHP
The
(summarized
hypoxic
to alleviate
coding
sequence of the genes
stop
fad
2x
(ATG) and stop codons (CTA/TTA) are
are given in italics. All oligonucleotides
direction.
55
written in bold letters
are
written in 5'
->
3'
Chapter
A PCR
2:
screening
Heterologous hemoglobin expression
method
used to
was
the correct gene inserts either in
pUC19
Boehringer Mannheim, Germany),
to
give
or
contained 0.4 ul DMSO, 1.0 ul 10X
sample
primer
identify
dNTP's
pmol/ul),
100
cell
growth
pKQV4.
Ta^-Buffer,
The reaction mixture for
0.1 ul
from
overnight-incubated
ampicillin (100 ug/ml)
techniques
a
PCR
for
amplified
concentration of each
primer (final
plate containing 96
wells
of
expected
Reaction mixtures
molecular
picked
with
was
analyzed
were
by
size
plasmid
and the correct recombinant
electrophoresis (33)
was
Standard PCR
plate.
well of the PCR
a
(Axon Lab,
plates (33) supplemented
agar
amplification (32).
fragments
gene
LB
and transferred into
used for DNA
were
one
Ta^-Polymerase (5U/ul;
Baden-Dättwil, Switzerland). A single, ampicillin resistant colony
aseptically
carrying
added to final concentration of 10 mM and H2O
well of
a
hypoxic
E. coli DH5a transformants
1 ul of each
were
total volume of 10 ul in
in
to alleviate
gel
agarose
used for further
characterization.
Plasmid DNA for DNA
grown bacteria cultures
The
Thermo
sequencing
using
sequencing
fluorescent-labelled
Sequenase
(Amersham International)
was
was
AAG GCG CAC TCC CGT
and -40 forward
and
of
or
-40
(IRD-41)
TCT)
or
reverse
to
pKKrev (5'
universal
automated
The
fragments
were
DNA sequence data
by using
cycle
collected and
software of LI-COR.
56
overnight
kit
sequencing
reactions
recommended
(34)
and
protocol
cycle
of
sequencing primers pKKfor (51
the
CTC
GAG TTC GGC ATG GGG TCA GGT
primers
were
a
from
(Qiagen, Switzerland).
for DNA
obtained
were
sequencing
from
separated
in
(LI-COR, Inc., Lincoln, Nebraska).
visualized
were
sequencing
sequencing reactions
DNA sequencer
Kit
primer
the
labelled
pUC19 derivatives, respectively,
(Ebersberg, Germany).
DNA
used for DNA
performed according
manufacturer. The infrared
G)
QIAprep Spin MiniPrep
a
purified
isolated and
were
scanning
laser
a
of
pKQV4
MWG-Biotech
LI-COR 4000 L
IRD-41
labelled
microscope assembly.
analyzed by using
the Base
ImaglR
Chapter
2:
Heterologous hemoglobin expression
Construction of VHb-reductase, FHP and
Plasmids
and
pRED2
pGE276
amplifications, respectively.
changed only
The gene
FAD domains of FHP
PCR
and
EcoRI
and
enzymes. Correct
of
(FHP),
and
reading
Construction of the
and
(VHb-NAD)
cases
expression
frames of the
were
gene
pGE276
as a
enzymes and the
encoding
NAD-domain
the
template
Pstl
digested
as
new
was
used
to
frame
construct
encoding
DNA
with
digested
with the
same
method and
pAXl (FHPg), pAX5
sequencing.
4 and
NAD
producing VHb-NAD
were
was
the
and
fragments
a
to
named
purified
was
cassette
PCR
digested
pUC19 digested
with the
pAX13.
PCR
The gene
3 and 7
fragments
fragment
(Table 1)
digestion
An identical
synthesizing
and
Hincll-
were
generate pAX2. The expression
generate pAX14.
expression
In all
isolated and
were
removed with EcoRI-Psfl
to
B).
6.5 kb Sall-Xhol
purified
amplified using oligonucleotides
pAX13
1
subunit of FHP
subcloned into
was
(Figure
necessary for the construction of the
(Table 1)
The PCR
plasmid
digested pKQV4
oligonucleotides
were
mentioned above. The
synthesizing FHPg-NAD
was
were
is summarized below
1 and 9
fragments
and subcloned into
into EcoRI-Psfl
by
they
1 and 3,1 and 2,
screening
cassettes in
fragment encoding FHPg
template.
with EcoRI and Hz'ncII. The
same
verified
pAX14 (FHPg-NAD)
amplified using oligonucleotides
same
expression
the PCR
that
FHPg and FHPg-
or
fragments
gene
between the
region
pKQV4 digested
using
so
flip
plasmids pAX4 (VHb-FAD-NAD), pAX9 (VHb-FAD), pAX12
cassettes. The
of
into
identified
subcloning steps using pUC19
two
fragment
Purified DNA
directly
were
pAX6 (FHPg-FAD)
linker
amplified using oligonucleotides
subcloned
plasmids
designed
were
fragments encoding FHP,
respectively (Table 1).
Pstl
authenticity
were
vectors.
for vhb and
templates
proposed
amino acid within the
one
protein domains (Figure 1).
and 1 and 10,
as
oligonucleotides
The
growth
FHPg-reductase expression
used
were
cell
hypoxic
to alleviate
cassette
and subcloned
cloning strategy
VHb-NAD
using
8, and 3 and 7 (Table 1) for the vhb and the open reading
domain
was
named
of
FHP,
pAX12.
57
respectively.
The
expression
vector
to alleviate
Chapter 2: Heterologous hemoglobin expression
hypoxic
cell
growth
146
A)
^VHb^
pPPCl
40
(^FHPg])PGGWK
pAX5
B)
LHIDVDA
FAD
NAD
147
CjHP^)
pAXl
4o;
C^Hb^)PGTWK
pAX4
LHIDVDA
FAD
NAD
258
dFHPg^PGGWK
pAX6
FAD
257
<^VHb^>PGTWK
pAX9
FAD
290
C!yHb^)fHIDVDi\
pAX12
NAD
Z91
t
CjHPg^)PHIDVD A
pAX14
Figure
(A)
1.
domains
Protein
of
flavohemoprotein (FHP). Alignment
three different modules in FHP:
and
NAD(P)H binding
the FAD-NAD
(LHIDVDA)
X13516 and X74334,
hinge
402)
sequences.
shorter
proteins
The linker
FHPg (147)
protein
pAX12 (VHb-NAD)
and
binding
FAD
between the
modules of chimeric
to Thrl49 in
and
regions
eutropha
R.
amino acid sequence reveals
(1-403)
FHPg (1-147),
and
domain
(153-258)
FHPg-FAD (PGGWK)
and
shown. The gene bank accession number for vhb and flip is
The
changed
domain
(VHb)
hemoglobin
and FHP
(1-146)
pAX4
and
proteins
pAX9 expressing
are
shown with the
VHb-FAD-NAD
and written in bold italics. VHb is
thus, the renumbering of
necessary. LHIDVDA linker sequence has
was
sequences in
are
Vitreoscilla
(1-257) proteins, respectively,
relative to
(146)
of VHb
(266-403).
domains
was
native
hemoglobin
respectively. (B)
Glyl50
VHb-FAD
or
domain
NAD
one
(1-
residue
amino acid residues in VHb fusion
to FHIDVDA
changed
pAX14 (FHPg-NAD), respectively,
and the
and PHIDVDA
changed
amino
acid residue is shown in bold italics.
Expression
constructed
were
gene
was
cassettes for VHb-FAD and VHb-F AD-NAD
was
a
slightly
modified
amplified using oligonucleotides 4
double
with the
using
digested
same
with EcoRI and
enzymes. The
plasmid
Kpnl
was
protocol
and 5
(Table 1).
pAX7.
1
B)
outlined above. The vhb
The vhb PCR
and inserted into
named
58
as
production (Figure
also
digested
reading
frame of
pUC19
The open
fragment
Chapter
the
flip
gene
(Table 1),
from
form
Heterologous hemoglobin expression
encoding
double
The
pAX16.
2:
digested
expression
with
An
identical
cassette for
cassette. The vhb and the
flip
cassettes
FAD), pAX12 (VHb-NAD)
sequencing
with
with EcoRI-Psfl
growth
6 and 10
pAX7, resulting
was
in
excised
digested pKQV4
for
applied
to
VHb-FAD-NAD-
fragment encoding
FAD-NAD
were
6, respectively (Table 1). The
VHb-FAD-NAD
was
named
The
pAX4.
pAX4 (VHb-FAD-NAD), pAX9 (VHb-
pAX14 (FHPg-NAD)
and the sequences of the
cell
VHb-FAD-expression
was
gene
in vectors
and
ligated
4 and 5 and 3 and
expression plasmid producing
amplified expression
ligated
cloning strategy
amplified using oligonucleotides
final
and Psfl and
EcoRI and Pstl and
hypoxic
amplified using oligonucleotides
was
Kpnl
complete expression
pAX16 using
pAX9.
FAD module
to alleviate
were
subjected
to
DNA
reading frames verified.
Bioreactor cultivations.
Cultures for both
DNA isolations and for bioreactor inoculations
plasmid
grown at 37°C, 250 rpm for
10 ml of LB medium
14 hours in 50 ml shake flasks
approximately
or
FHPg-reductase expression
batch medium
extract
(Difco)
supplemented
and 100
carrying
vectors,
with 150
mg/1
time. To start Sixfors bioreactor fed-batch
used to inoculate 300 ml of
H3PO4),
optical
glucose
were
densities
commenced with 1
casamino acids
and
using
in
ml/min
of air. The
of
a
defined
(Difco),
glucose
mg/1 yeast
30
Sixfors bioreactor unit
a
same
cultivations, 3.0 ml of seeding culture
(adjusted
by
performed
six controlled cultivations at the
7 ± 0.2
induced
VHb, VHb-reductase,
various
batch medium, and process
previously (19). Expression
constructions
the
pH
300 rpm, and 120
described
were
mg/1 ampicillin (19)
(Infors, Bottmingen, Switzerland) allowing
maintained at 37°C,
containing
(33) supplemented with ampicillin (100 ug/ml).
Fed-batch cultivations of E. coli MG1655,
FHPg
were
parameters
either with 2 M NaOH
composition
hemoglobins
IPTG-addition
(final
or
and
with 2 M
hemoglobin-reductase
concentration of 100
of cultures
ml/h
of feed medium when the culture reached
59
were
of feed medium has been
(A600)
were
was
approximately
uM)
when
1. Fed-batch mode
an
was
A600
of
Chapter
2:
Heterologous hemoglobin expression
approximately 2,
and the
approximately
Thereafter, the feeding
4.
feeding
rate
to alleviate
hypoxic
increased to 2
was
rate
cell
ml/h
growth
when
A600
maintained constant at 2
was
was
ml/h
until the end of 30 hours microaerobic fed-batch cultivations.
Dissolved oxygen concentration and exhaust gases
were
monitored
as
described
(CO2
and
from bioreactors
O2)
previously (17,19).
Analytical techniques
The soluble fractions of both
MG1655 cells
non-globin-producing
and
globin-expressing
E. coli
(for VHb, FHPg, FHPg-FAD-NAD, FHPg-FAD, FHPg-NAD,
VHb-
FAD-NAD, VHb-FAD, and VHb-NAD constructions)
100 ml of bioreactor cultivated cells followed
21M
were
centrifuge
by centrifugation using
pellets resuspended in 10
50 mM NaCl, 1 mM
(SLM-Aminco)
prepared by harvesting
at
1200
psi.
EDTA).
Cells
The cell debris
ml
were
were
lysis
buffer
(100
disrupted using
removed
tubes and clear soluble fractions
14000 rpm at 4°C in
reduced
+
an
Eppendorf centrifuge.
CO minus reduced difference
Acetate concentrations
SYNCHRON CX5CE
was
adapted
of bioreactor
recovered
spectroscopy
samples
with
a
Beckman
Beckman. The measured concentrations
60
French press
were
poured
described
in
15 min,
assayed using
previously (13).
Beckman
Mannheim Acetate Kit
(#148261)
and
was
by
used to
measure
Ethanol concentrations
autoanalyzer
the cultures.
Tris-HCl,
a
samples.
were
mM
a
were
determined
were
autoanalyzer system
from the bioreactor
enzymatically
as
Supernatants
by centrifugation
Globin activities
autoanalyzer. Boehringer
for the Beckman
acetate concentrations
measured
were
J2-
by centrifugation using
Beckman GS-6R at 4°C for 15 min, and 3200 g. The supernatants
new
Beckman
a
with rotor JLA 10.500, at 4°C for 10 min and 7.000 g.
discarded and cell
pH 7.5,
were
and
using
were
the alcohol kit of
normalized to the final Aôoo values of
Chapter
ß-lactamase
2:
Heterologous hemoglobin expression
activities encoded
the
by
to alleviate
hypoxic
cell
growth
plasmids pKQV4 (control), pAXl (FHPg),
pAX4 (VHb-FAD-NAD), pAX5 (FHP), pAX6 (FHPg-FAD), pAX9 (VHb-FAD),
pAX12 (VHb-NAD), pAX14 (FHPg-NAD)
using
the
chromogenic cephalosporin
(28).
The
cells
were
was
recorded at
was
determined
samples
Bradford
nitrocefin
pPPCl (VHb)
(Becton
withdrawn at the end of
were
disrupted using
a
&
Dickinson)
hypoxic
French press and the
change
determined
were
as a
substrate
bioreactor cultivations,
of absorbance at 482
nm
temperature (27). The total soluble protein of each sample
room
using
(5). Activity
and
Protein
a
Assay
ß-lactamase
of
is
based
(Bio-Rad)
Kit
reported
in
U/mg
FHPg)
and
the method of
on
of soluble
protein.
Results
Construction of
hemoglobin (VHb
and
hemoglobin-reductase
expression vectors
Most of the known bacterial
two different
activities,
an
two different biochemical
N-terminal
containing
VHb and the
reductase
recently
activity
Vitreoscilla.
catalyzing
E.
coli
contains
globin
VHb,
factor in
VHb-expression
such
if fusion of
as
a
a
an
has
reductase
(9, 46). However,
heterologous
a
been
protein
(Figure 1A).
not
it
is
have
well
function and
identified
in
system capable of
maintenance of the redox
heterologous reductase,
may be
an
host and decrease the beneficial effects of
hypoxic
conditions. In order
reductase domain to VHb
provides improved
oxygen stress under
heterologous
physiological
reductase
unidentified
catalyzed by
relieving
two-domain
of Vitreoscilla stercoraria do
for
These
activity (8, 43).
single
a
HMP, show
FHP and
polypeptide (16, 22, 44). However,
the reduction of VHb in vivo
activity-limiting
study
linked in
are
as
reductase
a
requires reductase activity
also
such
and C-terminal reductase modules
new
same
and
NADH-methemoglobin
state of heme iron of
to
properties
identified
in the
unlinked
an
oxygen-binding
hemoglobin
documented that VHb
such
hemoglobin proteins,
benefits, the following three expression
vectors
were
FAD-NAD), pAX9 (VHb-FAD), pAX12 (VHb-NAD).
61
constructed:
pAX4 (VHb-
The N-terminal domain of
Chapter
2:
Heterologous hemoglobin expression
highly homologous
FHP is
able to
expedite growth
hypothesis
we
of E. coli cells under
also constructed four
(FHP), pAX6 (FHPg-FAD),
plasmids
pPPCl
are
in
Figure
and
new
hinge
growth
possible
study
this
The constructions of these
VHb-expression plasmid
The structures of the novel
sequences, relative to the wild
that FHP is
pAXl (FHPg), pAX5
vectors:
pAX14 (FHPg-NAD).
cell
conditions. To
hypoxic
expression
previously (19).
hypoxic
it may also be
described in Materials and Methods. The
has been described
modules with
(8). Thus,
with VHb
to alleviate
type structures,
are
expression
summarized
IB.
Microaerobic bioreactor cultivations
14
i
i
i
i
I
i
i
i
'
'
'
12
1
10
15
Time
Figure
2.
Optical density trajectories
hemoglobin
domain
SixFors bioreactor
as
plasmid pKQV4 (T).
'
'
'
30
35
(h)
MG1655
expressing proteins showing FHPg
a common
culture conditions
The cells
were
cultivated under
hypoxic
conditions in
described in Materials and Methods.
VHb-expressing wild-type
relative to
coli
'
activity: pAXl (FHPg;»), pAX5 (FHP; A), pAX6 (FHPg-FAD;»), pAX14 (FHPg-
NAD; ) and the control
a
of E.
20
'
'
!
E. coli MG1655 cells showed
VHb-positive cloning
(19). Thus,
host E. coli DH5oc under
E. coli MG1655 cells
62
improved growth
expressing
VHb
behavior
oxygen-limited
(pPPCl), FHPg
Chapter
(pAXl),
and
or
were
cultivated at least twice in
dissolved oxygen concentration
after
approximately
end
of fed-batch
growth
microaerobic SixFors bioreactor. The
a
readings by Polarographie
Differences in
processes.
electrodes
were
zero
behavior
growth
between various
apparent after approximately 8-9 hours of cultivation, where
and
approximately 3,
MG1655:pKQV4
to
cell
6 hours of various cultivations and remained there until the
constructions became
was
hypoxic
separate globin-reductase fusions (pAX5, pAX6, pAX4, pAX9, pAX12
pAX14)
Aôoo
to alleviate
Heterologous hemoglobin expression
2:
ceased
the
of
growth
rapidly. Obviously,
the
non-VHb-expressing
supply
support effective growth of nonglobin-expressing
of oxygen
E. coli cells
was
control
not sufficient
beyond
this
point
(Figure 2).
14
i
i
i
i
i
i
i
i
i
i
i
i
i
10
Figure
3. Time
constructs:
NAD;
).
course
of
optical
i
i
i
i
i
>
15
20
Time
(h)
>
i
i
25
35
densities of E. coli MG1655 with the various VHb
pPPCl (VHb; .), pAX4 (VHb-FAD-NAD; A), pAX9 (VHb-FAD;*),
Data for cultivations of MG1655 cells
carrying
the control
and
hemoglobin
pAX14 (VHb-
plasmid pKQV4
are
marked
with(T).
The
growth
curves
of
MG1655:pAXl,
MG1655:pAX14 expressing FHPg
shown in
Figure
2.
The
or
growth
MG1655:pAX5,
MG1655:pAX6
FHP-reductase derivatives of A.
curves
63
of VHb-
(pPPCl)
or
eutrophus
and
are
VHb-reductase-
Chapter
2:
Heterologous hemoglobin expression
expressing (pAX4, pAX9,
and
pAX12)
to alleviate
E. coli MG1655 cells
Non-globin-expressing MG1655:pKQV4
used
was
of 8.3
of
(MG1655:pAX12)
coli
the
or
cells
NAD
approximately
optical density
(FHPg-FAD)
was
and
optical densities,
VHb-
or
cells
expressing
FHPg-expressing
3.
was
5.5 ± 0.5 at the end of 30
cells reached
final Aôoo
a
full
cells
MG1655:pKQV4 (5.5
domains reduced the final A6oo values
length
0.5)
±
an
(Figure 3).
A600
FHP-expressing
These results
was
construct
clearly
2 and
A.
on
plasmid pKQV4.
vectors
were
±
1.5)
reached
flavohemoprotein
cells reached the
clone
hypoxic
±
analyzed
method. The results obtained
75% and 15%
64
directly
can
its
be
higher
and the
0.5)
±
2 and
3).
improved
advantageous
activity.
for insert maintenance
using
0.5),
fed-batch cultivations
1.5), respectively (Figure
to combine
±
highest final optical
MG1655:pPPCl (7.9
fused reductase
also
relative to
MG1655:pAX5 (12.0
approximately
the average
a
slightly
VHb-expressing MG1655:pPPCl (7.9
MG1655:pAX5 (12.0
with
The
6.4 ± 0.9.
showed
respectively,
show that the beneficial effect of VHb
oxygen-binding properties
screening
FHP
slightly higher
MG1655:pAX14
E. coli
or
final cell densities relative to the control
higher
and
3).
eutrophus
substantially by using protein engineering
expression
the control
attain
MG1655:pAX9 (VHb-FAD)
original VHb-expressing
relative to the
eventually
of 13.9 ± 1.4 at the end of 30 hours
This value
MG1655:pPPCl (VHb)
6.4 ±1.0 and for
respectively. MG1655:pAX4 (VHb-FAD-NAD)
densities,
either
to
carrying
(Figures
VHb
with
together
reductase
9.0 ±1.0 and 9.9 ± 0.9,
2.2-fold and 50 %
approximately
the
relative
%
than the cultures
MG1655:pAX6
All
Figure
3).
but these constructions did
MG1655:pAX12
final
2 and
of
part
20
final A6oo for
improved
(Figures
FHPg (MG1655:pAX14)
MG1655:pAXl (FHPg),
final
shown in
are
0.9 which is similar to the final A6oo of 7.9 ± 0.5 for Vitreoscilla VHb-
+
Coexpression
E.
growth
internal control between
MG1655:pAXl (FHPg) expressing
expressing MG1655:pPPCl
of
cell
an
as
various cultivations and the final Aôoo of the control
hours cultivation. E. coli
hypoxic
different
by
a
PCR
oligonucleotide primer
Chapter
2:
Heterologous hemoglobin expression
combinations showed that the
prolonged hypoxic
Byproduct
are
a
accumulation during
needed to metabolize
assayed using
a
Beckman
hypoxic
such
products,
of reduction
surplus
cassettes
bioreactor cultivations
The excretion of side
relieve
expression
as
to alleviate
(data
not
were
acetate and
respectively.
were
autoanalyzer
from
and
samples
4.9
±
0.3
The
resulted
respectively)
in the
of
expression
MG1655:pAXl (FHPg;
mM/A600)
6.4 ± 0.3
in
a
the
± 0.4
or
(4.0
± 0.1
MG1655:pKQV4.
the smallest
4.6
was
mM/AöOO)
FHPg-expressing
mM/A^OO)
relative to
in
respectively,
MG1655:pAX6
relative to the
and
cells
± 1.5
produced
MG1655:pKQV4.
65
28%,
the end of
at
produced
2.9
strain
MG1655:pAX4, approximately
control
in
5.9 ± 0.7
and
These
40% less
mM/A^oo)times
more
MG1655:pAXl
under similar culture conditions. In addition, accumulation of ethanol
reduced
± 0.7
(Table 2).
samples
FHPg-producing
specific
either
22%
MG1655:pKQV4 (2.3
cells
specific
(8.2
MG1655:pPPCl (VHb;
(approximately
For
mM/A600,
0.1
alone,
domain
also measured from the
relative to
±
cultivation
of acetate relative to the control
Surprisingly, VHb-expressing MG1655:pPPCl
ethanol
in
VHb, FHPg and
% lower relative to
MG1655:pKQV4
decrease
cultivations. The results indicate that
(1.4
40
Our results
A600.
various
and
mM/A600
hemoglobin
mM/AJ
smaller
production
The excretion of ethanol
ethanol
unit of
one
expressing
approximately
were
were
withdrawn from bioreactors
MG1655:pAX5 (FHP),
acetate accumulation of the vector control
mM/A^oo)-
E. coli cells to
NAD+ and NADP+, which
constructions relative to the control
These values
during
Final concentrations of acetate and ethanol
glucose.
measured:
maintained
ethanol, allows
equivalents, regenerating
MG1655:pAX4 (VHb-FAD-NAD)
levels
growth
bioreactor cultivations
show reduced excretion of acetate from strains
acetate
stably
cell
shown).
at the end of 30 hours cultivation and normalized to
hemoglobin-reductase
hypoxic
25%
results
was
and
also
38%,
show that
Chapter
ethanol
2:
Heterologous hemoglobin expression
accumulation
relative to the
always
is not
hemoglobin-negative
decreased
in
cell
hypoxic
growth
hemoglobin-expressing
cultivated in
a
expressing various hemoglobin
hypoxic
constructions
bioreactor")
Plasmid
Hemoglobin
pPPCl
VHb
5.94 ± 0.69
pAXl
FHPg
6.43 ± 0.31
pAX4
VHb-FAD-NAD
4.89 ± 0.31
pAX5
FHP
4.63 ± 0.11
pAX9
VHb-FAD
5.63 ± 1.02
pAX6
FHPg-FAD
6.04 ± 0.96
pAX12
VHb-NAD
7.78 ± 1.41
pAX14
FHPg-NAD
6.38 ± 0.30
pQKV4
control
8.20 ± 0.69
Acetate concentrations
cells
control.
Table 2. Final acetate concentrations of E. coli MG1655
d)
to alleviate
Acetate
mM/A600b>
determined at the end of 30 hours of microaerobic cultivation and
were
normalized to A^„„.
oUU
b)
Mean
+
standard
Analysis
of
of the
error
biological
mean.
activities of VHb-,
hemoproteins by CO-binding
Cells
were
were
assays
using
the recorded
of
a
specific
qualitative, giving
soluble
activity
of the
technique (13).
of the heme-reductase-fusion
compare the
the
globins
on
proteins,
information about the
produced
the
results
it is
are
or
given
66
can
be
for
in Table 3. Due to lack
and to the novel
FHPg
impossible
specific activity
clearly
in all E. coli strains
globins (Table 3).
for
The maximum and minimum values of
to
predict
the effects
possible
activities of the different heme domains. Our results
no
were
in all constructions
the extinction coefficients. Thus, it is not
protein. However,
hemoproteins
samples
described in Materials
protocol
extinction coefficient either for FHP
of the reductase tails
globin-reductase
fed-batch cultivations and
spectra of the different constructions
reported
nature
standard
a
hypoxic
prepared following
and Methods. The biochemical
determined
and
assay
harvested at the end of
CO-binding
FHPg-
show
of
that
expressing
globins
are
to
only
per mg of
biologically
active
various recombinant
Chapter
Table 3.
2:
Heterologous hemoglobin expression to
CO-binding assay
Plasmid
a>
of various E. coli MG1655
alleviate
growth
cell
expressed hemoglobin proteins3'
minimum
Hemoglobin
hypoxic
[nm]
—
pPPCl
VHb
pAXl
FHPg
422
437
pAX4
VHb-FAD-NAD
422
443
pAX5
FHP
422
439
pAX9
VHb-FAD
419
436
pAX6
FHPg-FAD
421
438
pAX12
VHb-NAD
416
442
pAX14
FHPg-NAD
419
43(3
CO-binding assay performed according
The CO-difference visible
hemoglobin activity,
as
to
(13).
absorption spectrum
shown
of
MG1655:pKQV4
previously (19) indicating
revealed
that the recorded
represent the activity of overexpressed heterologous hemoproteins and
artifacts of E. coli
The
hemoglobin
absorption
(419
nm;
at
HMP-expression (data
domain
3).
and
verify
that FHP contains
VHb and also HMP of E. coli
of ß-lactamase
Production of
samples
a
or
has maximal
y-band
of VHb
FAD domain. A
changed
slight
in VHb-FAD-NAD-
strains relative to VHb
a
CO-binding fc-type
producing
slightly varying
domains of the fusion
at the end of
model recombinant
a
not
proteins.
three-
These
heme cofactor such
as
(30,41,43).
activity
withdrawn from
a
are
FHPg-NAD expressing strain was
maximum has
hemoglobin
curves
value is observed in strains
This variation may result from
dimensional structure of the
results also
for the
VHb-NAD-expressing (416 nm)
(419 nm) (Table 3).
Analysis
absorption
relative to the
fused either with FAD-NAD
absorption
no
shown).
eutrophus flavohemoprotein
absorption maximum (419 nm)
recorded. In addition, the
strain
of A.
A similar maximal
expressing FHPg protein
(422 nm)
not
slightly longer wavelength (422 nm)
a
Table
shift of the
(FHPg)
[nm]
maximum
419
hypoxic
cultivations.
protein, ß-lactamase,
microaerobic bioreactor
67
was
(Table 4).
also
assayed
from
The results showed
Chapter
that
MG1655:pAXl (FHPg;
MG1655:pKQV4 (88
20% and 35%
2.3-fold
3
higher
13
in the fastest
relative to either
U/mg)
U/mg), respectively,
production
U/mg).
± 3
The
growing
observation
various
optical
3
±
was
Final
relative
suprising
hemoprotein
62 ± 15
the
to
ß-lactamase
of
was
or
MG1655:pPPCl (VHb;
hypoxic
U/mg),
were
271
+
119 ±
fed-batch cultivations. The
40 ± 1
MG1655:pAX6 (FHPg-FAD;
and
MG1655:pAX14 (FHPg-
MG1655:pKQV4 (Table 4).
control
gene constructions
relative
3.1-fold and
because recombinant E. coli MG1655 strains
densities relative to the
Table 4. Final
a'
U/mg)
119 ± 13
MG1655:pAX4 (VHb-FAD-NAD;
reduced in
U/mg), MG1655:pAX12 (VHb-NAD;
NAD; 36
production
at the end of 30 hours
was
U/mg),
113 ± 10
ß-lactamase, respectively,
more
strain
growth
MG1655:pPPCl (VHb;
and
MG1655:pKQV4 (control)
ß-lactamase
of
U/mg)
106 ± 5
U/mg) produced 24%, 28%,
cell
hypoxic
U/mg), MG1655:pAX5 (FHP;
109 ± 3
MG1655:pAX9 (VHb-FAD;
to
to alleviate
Heterologous hemoglobin expression
2:
always
able to reach
This
expressing
higher
final
non-VHb-expressing control.
ß-lactamase activities of microaerobically grown
hemoglobins
E. coli MG1655
expressing various
ß-lactamase activity [U/mg]a>
Plasmid
Hemoglobin
pPPCl
VHb
119 ± 13
pAXl
FHPg
109 ± 3
pAX4
VHb-FAD-NAD
271 ± 3
pAX5
FHP
113 +10
pAX9
VHb-FAD
106 ± 5
pAX6
FHPg-FAD
40 ± 1
pAX12
VHb-NAD
62 ± 15
pAX14
FHPg-NAD
36 ± 3
pQKV4
control
ß-lactamase activity expressed
are given.
as
88
Units/mg
soluble
protein
+
3
is shown. The average and the
standard deviation
ß-lactamase
and
results
limitations
production
constructions
origin
of
here do not take into account the various
encountered
in E.
included the
reported
coli
same
were
with
the
(47). However,
each type of
genetical background
derived from the
replication. Thus,
optimization
protein production
here
with the
pBR322
parental plasmids
are
indicative and
able to redirect cellular
towards recombinant
are
experiment conducted
plasmid
the above results
cells
heterologous protein
of the E. coli MG1655 strain and
same
reductase-expressing
of
problems
relative to
68
suggest that
resources
more
FHP-expressing cells.
VHb-
efficiently
Chapter
2:
Heterologous hemoglobin expression
to alleviate
hypoxic
cell
growth
Discussion
Our results show that the beneficial effect of
bacterial
growth
be
can
improved substantially by expressing
effect of fused reductase
positive
protein
was
resulted in
a
2.2-fold
and
(FHP)
densities relative to the
2.5-fold
a
VHb-expressing
modulate cellular metabolism of E. coli
household
with the
by interacting
proton pumping, ATP production
nucleotide
cofactors
expression
elicits different
remain to be
changes
lacking
flip'
(24).
either in aerobic
the entire
strain.
a
a
physiological
in E. coli and in other
anaerobic
oifhp.
two
proteins
increase in final cell
has been shown to
VHb influences the energy
less reduced
increased
to
of
contingent
pyridine
been shown that HMP-
has
it
fusion
effects relative to VHb in E. coli. HMP is
growth
NO-related
species
functions of FHP of A.
heterologous
were
seen
gene in the genome
mutants did not accumulate nitrous oxide in
wild-type
of these
VHb-expression
against nitrosating agents,
flavohemoprotein
instead
respiratory chain; thus, leading
physiological
or
microaerobic
also observed when FHP
expression
(7,18, 27, 38-40).
rates and to
The detailed
investigated
was
(VHb-FAD-NAD)
(7, 18, 38, 39). Recently,
able to protect E. coli cells
oxidative stress
expression
in microaerobic E. coli. The
expressed
on
the vhb and the reductase gene module
protein (VHb-FAD-NAD) containing
The
VHb-expression
eutrophus
(8). However,
significant amounts
cells. This suggests that FHP may interact in
an
eutrophus
significant
hosts. No
in A.
A.
as
and
strains
eutrophus
observed in
unidentified way with
gas metabolism under denitrification conditions in A.
eutrophus (8).
The reductase domain
FHP is
part of the FNR-like
and
eucaryotic organisms
protein family
A well-examined
(1).
an
and is
N-terminal
module
(FAD-NAD)
widely spread
hemoglobin
consists
of
is the
example
two
of A.
eutrophus
among
procaryotic
flavohemoprotein (HMP)
domain and
a
of E. coli. HMP has
C-terminal FNR-like module
functionally inseparable
and
evolutionary
(43).
This
conserved
domains, the FAD- and NADP+-binding domains. The FNR-like module of HMP
reduces the heme-iron of the
hemoglobin
NAD(P)H
via the
(29)
to the heme
moiety
have shown that HMP is
a
domain
protein
by transferring
associated FAD group
reductase of broad substrate
69
electrons from
(1).
Poole et al.
specificity,
and the
Chapter 2: Heterologous hemoglobin expression
prosthetic
heme group of the
electron transfer in E.
electrons
domain is not
hypoxic
acceptors such
cytochrome
as
c
It may be
in
possible
or
via FAD to different
that FHP also functions in
a
growth
involved in the
able to donate
is
There
(29).
two different ways how electrons may be channeled from
FAD to the oxidized heme iron
cell
necessarily
Thus, the FAD-binding domain
coli.
to other
directly
hemoglobin
to alleviate
NAD(P)H;
either via
electron acceptors.
putative
similar way in A.
at least
are
eutrophus
and
even
heterologous prokaryotes.
Normally,
binding
the
of oxygen
group of the
[Fe(III)]
binding
lead to
can
of
physiological
binding
effects of the
reductase
activity capable
negligible
rate
Previously,
of
an
has
it
constitute
cytochrome P450,
an
monooxygenase
metabolic
an
reductase
in
of
activity
a
in
S.
tropicalis
led to
cerevisiae.
uptake
(35).
an
novel
host and
hemoglobin-like
regeneration
domain.
The
electron
expressed
in
indirectly suggest
an
of
NADPH
a
as
NADPH
cytochrome
catalysed
shows that the
VHb, may be limited by
flow
may
NAD(P)H
an
to the heme of the
concomitantly
prosthetic
increase
the
heme of oxygen
E. coli.
All constructions showed VHb and
Our results
a
experiment
rate of the ferriheme to ferroheme of the
binding hemoglobin in
for the
suggests that the coexpression of
additional reductase may increase electron flux from
0
several fold in Vitreoscilla
coexpression
such
a
(11, 29).
NADH-cytochrome
elevated level of P450
This
heterologous hemeprotein,
a
ferric citrate with
electron-transferring path
alkane inducible monooxygenase, with
activity
activity
called
protein,
been shown that the
P450 oxidoreductase of Candida
as
reductase activities in E. coZf
associated reductase
also
such
complexes
iron
oxidation of NADH and to increase the oxygen
(46). Recently,
the biochemical and
HMP has been shown to possess flavin
ferric/flavin
to
limiting
[Fe(II)]
The oxidized iron
[Fe(III)] (26).
oxygen, therefore
reducing
reductase, have been shown
reversible reaction, but the
a
redox reaction, in which the ferroheme
hemoglobins.
relative to main
VHb and
a
is oxidized to ferriheme
hemoglobin
incapable
is
of O2 to the heme iron is
FHPg activity
as
judged by CO-binding
that the recombinant reductase
active form because the fusion constructions
70
system
assay.
was
expressing
also
either
Chapter
Heterologous hemoglobin expression
2:
VHb-FAD, FHPg-FAD, VHb-reductase and
relative to either VHbis
supported by
or
results
FHPg-expressing
showing that
H+ ions from NADH to FAD and
via the heme group to
reduction
the
equivalents
electron
could not be
from
constructions. As
proteins
is
endogenous
after the oxidation of the
beneficial effect of the
were
E. coli
cell
hypoxic
growth
able to increase cell
(Figure
2 and
This
3).
growth
assumption
the native enzyme of E. coli HMP transfers two
substrate
passed
FAD
passes the electrons in two steps,
(29).
Therefore it is
the
or
that either
novel
three
dimensional
able to hinder the transfer of reduction
reductase domain
hemoglobin
prosthetic
likely
from NADH to the heme due to lack of
moiety
sterically
consequence,
a
FHP
consecutively
putative
transfer-mediating
structure of fusion
equivalents
a
to alleviate
to
VHb-NAD and
has reduced
affinity
heme group of the heme
heterologous hemoglobin expression
FHPg-NAD
towards oxygen
moiety. Thus,
is lost in
hypoxic
the
E. coli.
Acknowledgements
This research
supported by
was
for skillful DNA
the ETH. We would like to thank Ms. Heidi Ernst
sequencing of the plasmid
constructions.
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Heterologous hemoglobin expression
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protein related
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Chapter
3: Metabolism of
hypoxic hemoglobin-expressing
E. coli
Dissection of the central carbon metabolism of
expressing Escherichia
analysis
coli
by
13C NMR flux distribution
in microaerobic
75
hemoglobin-
bioprocesses
Chapter
3: Metabolism of
E. coli
hypoxic hemoglobin-expressing
Abstract
Escherichia coli MG1655 cells
expressing
eutropha flavohemoprotein (FHP),
(FHPg)
and
fusion
domain
(VHb-Red)
a
protein
were
labeling
NMR
of the
proteinogenic
spectroscopy. The
fluxes between E. coli
eutropha
FHP
either VHb
on
or
or
the N-terminal
of VHb and the R.
grown in
low oxygen concentrations
Vitreoscilla
hemoglobin (VHb),
domain of FHP
hemoglobin
C-terminal reductase
eutropha
microaerobic bioreactor to
a
the central carbon metabolism,
amino acids and two dimensional
expressing
VHb-Red
using
running cyclically,
Consistently,
production
E.
of the
implications
coli
were
cells
were
either VHb
or
VHb-Red and cells
(FHPg).
met
cells
a
by
a
increased flux
shift
towards
FHP
and
a
of these observations for
aerobic
cycle
In
is
regulation.
showed
lower
acetate and D-lactate. The
biotechnological applications
76
in VHb- and
the TCA
FHPg activity
by-products formate,
cycle.
through glycolysis.
more
R.
expressing
branched TCA
eutropha hemeproteins
R.
displaying
anaerobic
an
expressing
E. coli MG1655
found to function with
expressing
indicating
typical
fractional re¬
[13C, 1H]-correlation
Furthermore, cellular demands for ATP and reduction equivalents
contrast, in E. coli cells
the effects of
study
NMR data revealed differences in the intracellular carbon
the truncated heme-domain
VHb-Red-expressing
Ralstonia
are
discussed.
Chapter
3: Metabolism of
hypoxic hemoglobin-expressing
E. coli
Introduction
Hemoglobins
are a
specific
group of
that
oxygen-binding proteins
be found in
can
mammals, plants and microorganisms (15). The homodimeric hemoglobin of
Vitreoscilla
is the best-characterized bacterial
VHb is up
(VHb)
oxygen limitation
regulated by
physiological
functions
have
heterologous expression
of
hemoglobin.
The
expression
in Vitreoscilla
(hypoxia)
(50),
VHb
has
been
used
physiologically
alleviate
to
but its
Nonetheless,
yet been entirely elucidated.
not
unfavorable effects of oxygen limitation and to
improve growth properties
productivity
and mammalian cells that
of various
microorganisms, plants
of
and
yield
industrially important metabolites (7,18, 21, 28-30, 32, 35).
Previous research
mainly
focused
coli cells
on
lacking
the effects
on
either
(in cydr, cyo+ strain)
complexes
relative to
from
heterologous
operation of the respiratory
the
cytochrome
complexes (microaerobic
o
arising
terminal
and
a
(aerobic
o
terminal
revealed
oxidase)
1.5-fold increase in
wild-type
cells
(48).
chain.
a
VHb
expression
of VHb in E.
Expression
oxidase)
or
cytochrome
5-fold increase in
d
cytochrome
has
d
cytochrome
(in cyd+,
These results have led to the
cyo-
strain)
hypothesis
that VHb is able to increase the effective intracellular oxygen concentration with
concomitant increase of the amount of
translocation
activity
ratio relative to
gradient
to
across
generate
have
a
30%
a
fold lower in
larger proton
complexes
complexes
(16).
i.
e.,
complexes (20):
is characterized
activity
cells
(25, 36, 38). VHb-expressing cells
and
a
reported
65%
higher
a
a
the
proton
higher H/O
larger proton
indeed able
are
that the
are
in
a
more
is consumed
more
(8,20).
ATP turnover rate
steady
state
level is 1.8-
NAD(P)H
grown under
nearly
anoxic
reduced state under oxygen limited
Anoxia is known to reduce electron flow
NAD(P)H
by
and is able to generate
VHb-expressing strain than in control cells
conditions
that
ATPase
o
o
flux per reduced oxygen molecule than control cells and
it has also been
(47),
so
d
the cell membrane
conditions
chain,
cytochrome
cytochrome
higher
Furthermore,
of
cytochrome
through
slowly. Therefore,
one
the
may
respiratory
hypothesize
that under low oxygen tension the presence of VHb in E. coli increases the electron
77
Chapter
flux
through
shift in the
3: Metabolism of
hypoxic hemoglobin-expressing
respiratory chain. Moreover,
the
NAD+/NADH
concentration ratio
Tsai et al.
VHb-expressing
flux
and
positive
cells direct
pathway (PPP)
(TCA)
than
generates
excess
E. coZz
creates
the pentose
phosphate
tricarboxylic
acid
Direction of additional
glucose through
PPP
transhydrogenation
a
H+-flux from NADPH to NAD+.
an
cycle
cells
VHb-expressing
reduced formate and D-lactate excretion levels relative to
(49).
Recently,
we
constructed
novel
a
set
engineered hemoglobin proteins (12).
flavohemoprotein
of R.
and contains
redox-active
(Red)
an
high
and
hemoglobins
growth rates
sequence
was
in
a
VHb and the
was
and
a
C-terminal
fused with VHb to
addition, the hemoglobin domain (FHPg),
in
was
of metabolic
truncated from the reductase
coli.
E.
interesting features,
activity
were
and
native
member of the FNR-like
hemoglobin (FHPg)
functionally expressed
changes
FHP is
homology with VHb,
showed various
and
In
used
proteins
The redox-active domain
domain.
for
expression systems
The native
N-terminal
generate the VHb-Red fusion protein.
which shares
of
eutropha (FHP) (9).
proteins (23)
domain
that VHb-
of NADPH, and results in
amount
displayed strongly
controls
(49).
the
key steps
suggested
glucose through
acetyl-CoA through
on
Therefore, metabolic
E. coli.
metabolic model
a
fraction of
higher
and channel less
reaction, which
also
a
wild-type
an
performed
was
that this
(47) postulated
might have implications
of the central carbon metabolism in
analysis (MFA)
E. coli
such
The
as
pathways
expression
of
the
significantly improved
under
hypoxia (12).
The ratios of intracellular metabolic fluxes in the central carbon metabolism of E.
coli MG1655 cells grown under
determined
and 2D
that
using biosynthetically-directed
[13C, 1H]-correlation
serves as a
reference for the
under
oxygen-limited
microaerobic
NMR
presently
studied
conditions
pyruvate-formate lyase
activity.
and the
fractional
previously
13C-labeling
been
of amino acids
spectroscopy (11). This investigation, which
the
characteristic of anaerobic metabolism,
of the
conditions have
hemoglobin-expressing cells,
topology
as was
primarily
suppression
This result is at variance with
previously
78
of
of
the
active
evidenced
showed
pathways
by
the
is
activity
2-oxoglutarate dehydrogenase
assumed aerobic
configurations
Chapter
and illustrates the
active
3: Metabolism of
importance
of
hypoxic hemoglobin-expressing
E. coli
characterization of the
experimental
topology
of
pathways.
Further studies of the central carbon metabolism of
promise
to lead to
into the cellular
insights
new
support the design of improved
applied
the
labeling
of
proteinogenic
carbon flux ratios
using
a
amino acids
variety
of
physiology,
of
(43-46)
cells
which in turn may
biotechnology. Therefore,
strains for
recently developed approach
hemoglobin-expressing
biosynthetically-directed
we
now
fractional
for the assessment of intracellular
E. coli strains.
hemoglobin-expressing
Materials and Methods
Strains and
The
plasmids
plasmids pPPCl, carrying
eutropha
hemoglobin
flavohemoglobin
of R.
(flip)
(fhpg),
domain
gene
gene
the Vitreoscilla vhb gene,
pAX5,
and
eutropha
[k
coli MG1655
plasmids
used to
were
has been described
Spring
Harbor
Laboratory].
the
the R.
native
the gene fusion
coding sequence
consequences of
study physiological
F ; Cold
,
containing
pAX4, carrying
between the vhb gene and the FHP reductase domain
(vhb-red)
pAXl, encoding
of R.
eutropha
globin expression
in E.
The construction of the
previously (12,22).
Microaerobic bioreactor cultivations
Cultivations of E. coZz MG1655
(concentration
of
Switzerland)
a
in
the sole carbon
O2
genes
reduced
2
g/1
performed
mmol/1)
using
in
a
(fhpg
and
for
these
flip),
the
strains.
a
under microaerobic conditions
SixFors
Based
on
glucose
This
(Infors,
unit
containing glucose
previous experiments,
for E. coli cells
79
bioreactor
minimal medium
(4 g/1 glucose) (22).
glucose consumption
hemoglobin
to
0.02
fed-batch mode
source
showed smaller
=
were
expressing
the R.
which
eutropha
concentration of the medium
modification prevents
both
as
was
glucose
Chapter 3:
accumulation and
bioreactor
dilution
parameters
temperature 37°C,
Metabolism of
of
Inocula
media
were
aeration rate 120
a
a
13C-substrate
ml/min of
of 0.2. The
ml/hour
mixture of 10%
O2
the various
monitor
(Brüel
At Aöoo
and CO2
&
=
2.5, and
started at Aöoo
uniformly
glucose.
consumption
Aöoo
at
13C-labeling
unlabeled
labeled
=
production
Kjaer,
was
phase (A000
determined at the
=
of the
biological activity
CO-binding activity
with
containing
37°C and 250 rpm.
at
was
starting absorption
a
induced with IPTG to
of the cultures
increased to 2
4.5. The
labeling
10 ml of LB-
was
started with
ml/hour
when the
medium contained
were
were
monitored
Type 3427)
controlled with
of the
at 600
and 90%
harvested for NMR measurements.
of the cells
Growth
a
nm
cultures
a
and
using
dissolved
Polarographie
was
=
4.5)
oxygen
electrode
monitored with
every 30 minutes. Cellular
beginning (Aöoo
emission
an
a
dry weight
and the end of the
labeling
expressed hemoglobin proteins
of the
assay
analysis
By-product
concentrations
SYNCHRON CX5CE
of
expressed hemoglobin proteins
by-products
were
autoanalyzer
concentrations
was
confirmed
by
a
(17).
Quantitative
glucose
adjusted
7) (40).
Biological activity
The
Feeding
was
=
tightly
spectrophotometer (Perkin Elmer)
was
hemoglobins
Emission Monitor
(Mettler-Toledo, Switzerland).
7.0 ± 0.2,
13C-glucose (Isotech, Miamisburg, OH)
7 the cells
concentration of the cultures
(CDW)
pH
standardized to obtain
was
final concentration of 0.5 mM at Aöoo «1.
fractional
air and
ampicillin (100 fxg/ml)
with
expression of
The
glucose.
ml, stirrer speed 300 rpm,
grown for 14 hours in 50 ml shake flasks
(39) supplemented
rate of 1
unlabeled
with
volume 300
E. coli
2 M H3PO4.
or
Inoculation of the bioreactors
(Aôoo)
the
working
were:
addition of either 2 M NaOH
hypoxic hemoglobin-expressing
were
and intracellular metabolites
measured
at
enzymatically
with
a
Beckman
one-hour intervals. Ethanol and residual
determined
80
using
Beckman alcohol
(#445900)
and
Chapter
3: Metabolism of
glucose (#442640) kits, respectively.
Roche
concentrations
were
The acetate kit
Quantifications
Diagnostics.
E. coli
hypoxic hemoglobin-expressing
(#148261)
D-lactate,
of
performed using enzymatic
purchased
was
for the Beckman
adapted
assays
formate
and
succinate
from
autoanalyzer system (5,49).
Quantification of the intracellular metabolites pyruvate,
performed using perchloric
concentrations
According
to
to the
were
Beckman
a
obtained
from
supplier information, unspecific
measured concentrations, which
the
and
for extraction of the metabolites
(PCA)
measured with
were
(5, 10). Enzymes
assays
acid
ATP
was
(2,10).
The
autoanalyzer using enzymatic
Roche
Molecular
Biochemicals.
reactions contribute less than 1 %
verified
we
ADP
experimentally (data
not
shown).
Sample preparation
Fractionally
and 2D [13C, 1Hl-COSY NMR measurements
labeled cells
spectra of the amino acid
frequency
detected
[13C, 1H]
recorded for each
using
during
delay
a
and the
Varian
between
scans)
gradients
timax
with the carrier
=
412 ms;
frequency
width of 33.8 ppm. The spectra for the aromatic
hours
(950x512 complex points;
between
scans)
of 22.8 ppm.
with the 13C-carrier
Spectra
resolution after
for the
timax
were
zero-filling
was
0.9
2
respectively,
412ms;
frequency
transformed
spectrum with the aliphatic
Hz/point along
=
using
t2max
=
=
(42)
was
128 ms; 1.5
s
relaxation
a
spectral
recorded in 7.5
109 ms; 1.5s relaxation
spectral
a
the program PROSA
(14).
for the spectra
applied
recorded in
ppm and
set to 123.8 ppm and
resonances,
were
used for coherence
resonances were
t2max
resonance
spectra
resonances were
set to 42.5
Hz/point along
81
were
scheme WALTZ
aliphatic
the
proton
coherence
single quantum
containing
a
spectrometer. Two proton-
Inova 400
decoupling
(1400x256 complex points;
110°C, and the NMR
recorded at 40°C and at
Pulsed field
sample (6).
detection. The spectra
12.5 hours
were
heteronuclear
pathway rejection (4, 52)
in 6N HCl at
hydrolyzed
mixtures
of 400 MHz,
2D
were
ooi
and 0.6
and 2.0
The
width
digital
Hz/point along
Hz/point along
containing
delay
the aromatic
an
02
and 4.6
resonances.
Chapter 3:
The overall
1H-NMR
degree
Metabolism of
of the 13C
spectra (timax
hypoxic hemoglobin-expressing
labeling
of the amino acids, Pi,
1.024s; 8s interscan delay),
=
as
E. coli
measured from ID
was
shown in
Figure
1.
ta)
1000
I
1
'
rT5
JlIl
'
7*
'"/
'
ÎTT
TT"
24
r
L0(lH) W""
1. Determination of the
spectrum, (a) Region of the
the aromatic
resonances.
Expansion showing
protons bound
sum
of the
resulting
fraction of
of overall 13C
degree
peaks
corresponding
value of Pi
(4.5%
was
[13Cé]-glucose
in this
in
of the biomass from
The satellite doublet
12C-H
peak
are
indicated
integral
ß (43).
42
arrows.
eight principal
according
to
arising
(b)
from
The ratio of the
peaks yields
the overall
case).
good agreement with
the value calculated from the
13C-13C
analysis
of the scalar
coupling
fine
(40).
The value for Pi
coupling
structures
were
analyzed
the program FCAL
82
(46).
in
the
fragments present
intermediates of the central carbon metabolism
Szyperski (43), using
was
fine structure of Leu-
correlation spectra, and the relative abundance of intact carbon
in
ID !H-NMR
in the minimal medium and the fraction of 13C-labeled
confirmed from
individual
by
of all three
biomass assessed via first-order wash-out kinetics
independently
a
allow observation of the 13C satellites,
(unassigned).
resonance
of the two satellites to the total
of the biomass
labeling
spectrum recorded for the hydrolyzed biomass comprising
Several well-resolved
well-resolved
ppm
V-fl
c-n
ID XH-NMR
to 13C and the
integrals
labeling degree
The
a
5 45
t
t
V-h
Figure
'
57,5
were
Flux ratios
derived
through
Chapter 3:
several
key pathways
the abundances of
Metabolism of
hypoxic hemoglobin-expressing
in the cellular central metabolism
fragments
as
described
E. coli
then calculated from
were
previously (40,43).
Results
Physiological
characterization of the
The E. coli strains
MG1655:pPPCl, MG1655:pAXl, MG1655:pAX4
pAX5 displayed CO-binding
biochemically
hemoglobin-expressing strains
activities that
hemoglobin proteins.
active
hemoglobins corresponded
to the values
8
'
I
indicative for the
are
'
expression
absorption spectra
The
reported previously (data
'
and MG1655:
l"l—1—1—|—T—1
1
1
7
*
6
°mm
1
1
1
1
of
of the various
not
shown) (12).
1
3*
:
5
4
o
X
3
o
•
°*<
2
-
O
•
o*
.
•
1
s
,
-
•
»,,,,
1
.
,
.
5
10
Time
Figure
2. Growth
study,
when
proteins
were
was
trajectories
growing
in
induced at Aôoo
harvested for NMR
expressing
expressing
a
the VHb
the
of the four
FHPg protein;
hemoglobin-expressing
1 and the fractional
analysis
protein;
(h)
minimal medium under
=
•
x,
,
15
at the end of the
hypoxic
13C-labeling
conditions. The
was
MG1655:pAX4, expressing
expression
started at Aöoo
cultivation, with Aôoo
MG1655:pAX5, expressing
83
E. coli MG1655 strains used for this
the FHP
the VHb-Red
=
7.
protein;
protein.
of the
4.5. The cells
MG1655:pPPCl,
,
o
~
,
MG1655:pAXl,
Chapter 3:
Metabolism of
The MG1655 strains that coexpress
hypoxic hemoglobin-expressing
and reductase activities, FHP and
hemoglobin
VHb-Red, grew fastest, exhibiting specific growth
The E. coli strains
respectively.
h"1 and |ii
7
=
0.148 lr1,
expressing
either
E. coli
rates
FHPg
of 0.168 lr1 and 0.180 h-1,
(u)
VHb grew with
or
respectively, during the 13C-labeling phase from
u
Aöoo 4.5
0.132
=
to
Aöoo
(Figure 2).
Glucose
and the
uptake
production
of D-lactate, acetate, ethanol and succinate
calculated and normalized to CDW to obtain the
mMol/h/g) (Table 1).
labeling phase
assayed
Evaluation of qp for
revealed
slight
a
course
a
global
view
We observed
R.
eutropha
over
the whole
substantially
FHP
FHPg (MG1655:pAXl;
mMol/h/g)
and
1.30
VHb-Red
0.14
expressing strains,
production.
strains
The
approximately
Furthermore,
we
for the strains
found
acetate
an
eutropha
FHP- and
by
the NMR
the
rates of all
analysis represent
or
for the strains
the truncated
relative to VHb
3.31
rates
acetate
were
FHPg-expressing
into the
2-fold
VHb and VHb-Red
84
2.59
FHPg
for
acetate
similar for the MG1655
expressing
culture media
VHb-Red
(Table 1).
higher glucose consumption
(3.97
strains
and 4.60
(2.32
highest glucose consumption
(Table 1).
and
FHP
observed
was
and FHP, whereas the strain
approximately
flavohemoglobin
(MG1655:pPPCl;
mMol/h/g).
reduction
production
more
expressing
relative to the VHb- and VHb-Red-
production
pronounced
2-fold
expressing
control strain showed
CDW
less
specific
mMol/h/g)
(MG1655:pAX4;
expressing VHb, FHPg
excreted
the R.
and
specific production
production rates
mMol/h/g)
showed also decreased D-lactate
during
(qp;
labeling phase.
lower formate
(MG1655:pAX5;
rates
of the cultivation. We did not take into
account these variations since the values assessed
also
time intervals
subsequent
decrease in the
metabolites within the time
specific production
were
mMol/h/g)
and 2.88
rate
being
relative to
mMol/h/g).
5.45
rate
The
mMol/h/g
Chapter
Table 1.
Physiological
3: Metabolism of
hypoxic hemoglobin-expressing
data of the five E. coli strains
MG1655:pPPCl (VHb),
MG1655:
E. coli
MG1655:pAXl (FHPg), MG1655:pAX5 (FHP),
pAX4 (VHb-Red)
and MG1655
(control)
assessed
during re¬
labeling phase.
Parameter
FHPg
FHP
VHb
VHb-Red
control3)
'
2.88
2.32
3.97
4.60
5.45
qethanolb)
0.53
0.09
0.53
1.02
0.32
2.68
2.14
2.27
4.57
3.05
1.30
0.14
2.59
3.31
4.10
qD-lactateb)
0.13
0.87
1.43
1.14
0.76
q succinate
0.22
0.25
0.58
1.92
0.51
4.18
5.59
2.74
3.19
1.78
3.50
5.19
1.27
2.18
0.78
0.48
0.69
0.43
qglucose
'
qacetate
qformate
'
'
qo2b)
qco2b)
RQc)
0.84
0.95
Ud)
0.132
0.168
0.148
0.180
0.125
YX/Glce)
0.258
0.249
0.176
0.159
0.143
a>
Non
b)
The rates
hemoglobin expressing E.
are
coli MG1655 strain
given in mmol/ h/ g cdw.
c>
Respiratory quotient calculated from O2 uptake and CO2 production rates.
d'
Specific growth rate in h4
e> Growth
yield on glucose in g biomass / g glucose consumed
Monitoring
the exhaust gases
consumption (qÛ2)
control strain,
The
rates. The strains
evidenced
as
by
respiratory quotient, RQ
of the cells. The calculated
were
0.84 and 0.95,
strains
expression
and 80%
of R.
eutropha
production (qC02)
FHP and
VHb-Red
FHPg
an
revealed
expressing strains,
higher qC02
is
and O2
and
q02
values
higher
or
the
(Table
indicator for the metabolic state
RQ values for MG1655:pAXl and for MG1655:pAX5
whereas those for VHb-
lower
FHP
or
being
FHPg
(MG1655:pAXl), respectively,
Vitreoscilla
0.48
and
0.69,
increased the RQ
relative to
or
VHb-Red-expressing
respectively. Thus,
by
100%
MG1655:pPPCl
the
(MG1655:pAX5)
strain
expressing
hemoglobin (Table 1).
Interestingly,
we
for the strains
R.
qC02/ q02,
=
respectively,
significantly
were
or
at least 2-fold
an
CO2
expressing
activities than either the VHb-
respiratory
2).
yielded specific
could
assess a
expressing
2-fold
Vitreoscilla
higher
intracellular pyruvate concentration
hemoglobin
relative to the strains
eutropha hemoglobin proteins (Table 2). Pyruvate
breakdown in the
glycolysis,
consumed in the anaerobic
is the
possessing
of
endproduct
and is further channeled towards the TCA
pathways leading
85
to the formation of the
glucose
cycle
or
is
typical by-
Chapter 3:
products
of anaerobic
concentration
pathway
was
Metabolism of
in
be
can
growth. Thus,
explained by
MG1655:pPPCl
identical with the VHb-
analysis
and
or
a
this
difference
higher glucose
MG1655:pAX4
approximately
2-fold lower
flow
through
ATP/ADP
under
pyruvate
glycolytic
of pyruvate
On the other
significant
study,
the
production
VHb-Red-expressing strains.
hemoglobin-expressing strains
E. coli
intracellular
in
cells. The
of ATP and ADP contents did not reveal
any
the different
strains
hypoxic hemoglobin-expressing
hand, the
differences among
but the control showed
ratio relative to the
an
hemoglobin-expressing
(Table 2).
Table 2. Concentration of intracellular metabolites
pyruvate,
ATP and ADP in E. coli strains
MG1655:pAXl (FHPg), MG1655:pAX5 (FHP), MG1655:pPPCl (VHb),
and MG1655
Metabolite3)
MG1655:
pAX4 (VHb-Red)
(control)
FHPg
FHP
VHb
VHb-Red
controlb)
6.2 ±2.7
6.7 ±2.6
11.7 ±7.3
12.4 ±3.9
12.2 ±1.4
ATP
46.7 ± 10.5
33.9 ± 5.3
37.3 ± 9.2
38.5 ± 2.4
22.8 ± 7.6
ADP
12.9 ± 8.8
9.1 ±3.8
10.4 ± 3.2
15.6 ±4.7
15.6 ±1.8
3.6 ±0.2
3.7 ±0.1
2.5 ±0.1
1.4 ± 0.2
Pyruvate
ATP/ADP
a> For
each metabolite four
determined
as
independent samples
Validation of the
performed
taking
was
closed to within
a
analysis
for all
glucose uptake, production
larger
value and the standard deviation
are
data
carbon balance
by-products.
C-uptake
mean
E. coli MG1655 strain.
physiological
into account the
excretion of
and
a
± 0.1
have been taken and the concentrations have been
described in Material and Methods. The
given in umol/g CDW.
b> Non
hemoglobin expressing
We
3.6
hemoglobin-expressing
strains,
of biomass, CO2 evolution and
The carbon recovery calculated
as
the ratio of
C-output
than 93% in all cases, i. e., the carbon balance could be
few percent
(Table 3).
86
Chapter
3: Metabolism of
hypoxic hemoglobin-expressing
Table 3. Carbon balance for the validation of the
MG1655:pAX5 (FHP), MG1655:pPPCl (VHb),
physiological
MG1655:
data in
E. coli
MG1655:pAXl (FHPg),
pAX4 (VHb-Red)
and MG1655
(control)»)
Metabolite
FHPg
FHP
VHb
VHb-Red
Controlb)
Glucose
112.4
88.7
192.7
196.3
166.7
Acetate
35.8
24.6
70.2
40.3
41.5
Ethanol
6.9
1.6
14.2
12.1
13.8
Formate
9.2
0.9
23.8
19.6
26.8
D-lactate
2.7
0.2
24.0
20.7
35.2
Succinate
2.7
6.5
24.0
20.7
20.1
C02
12.3
19.5
7.6
4.9
4.0
Biomass
34.6
33.0
33.0
41.4
31.0
104.2
86.3
196.0
184.4
172.5
93
97
102
94
103
Carbonout
%
Recovery
a) Carbon
period.
balances
Values
biomass
are
were
calculated from the concentrations measured in the media
given
in mmol carbon.
Cout: summation
over
standard content of 39.8 mmol carbon per 1 g CDW
b) Non
hemoglobin expressing E. coli MG1655 strain.
a
Analysis
The
of the 2D f13C, 1Hl-COSY
13C-labeling experiments
metabolism when
cells
observed for
anaerobically
MG1655 cells
(11),
fluxome
The
was
(Fig.
labeling pattern
revealed
that
OAA
building
3
indicating
the
labeling
outputs.
For the
assumed.
and
FHPg-/FHP-expressing
and
similar to those
E. coli cells
wild-type
FHPg-
(43)
FHP-expressing
cells.
previously
and microaerobic
cells
a
more
aerobic
/ Table 4).
solely
is
blocks
strains
respectively,
expressing
were
the
in
a
branched fashion to
(Table
either FHP
generated through
activity
cycle
also
of the
87
cells
phosphoenolpyruvate (PEP) by
biosynthesis
that in these cells the TCA
respiration. Concomitantly,
from
cycle operates
for
VHb-/VHb-Red-expressing
of
(OAA)
derived
the TCA
microaerobically growing
88% of the OAA,
grown
over
differences in the central carbon
major
yielded labeling patterns
of oxaloacetate
carboxylation (33). Thus,
exclusively
revealed
whereas for
found
was
assayed
carbon
spectra and derivation of metabolic flux ratios
comparing VHb-/VHb-Red-
VHb/VHb-Red-positive
all
serves
4).
or
the
In
generate
contrast,
FHPg, only
for
44% and
anaplerotic reaction,
for ATP
production
via
pyruvate-formate lyase (Pfl) (26)
is
Chapter
suppressed
3: Metabolism of
is activated. Further
respiration
as
ratios for the interconversion of
strains
expressing
VHb
expressing FHPg (0.19).
is
compatible
production
Table 4.
with
our
rate for the
Origin
(0.73)
In
pyruvate
analysis
and VHb-Red
physiological
data
E. coli
showed increased turnover
to AcCoA and formate
(0.65)
MG1655:pAX5 (FHP)
Pfl
and to
a
activity
displaying
a
FHP-expressing strain (Figure 3,
FHPg
Pentose
pentoses (ub)c>
FHP
by
Pfl for the
smaller extent for cells
detectable, which
is not
very small
Table
specific
formate
4).
of intermediate metabolites in the four cultures of Table 1 and
Metabolite
PEP from
hypoxic hemoglobin-expressing
a
control cultivation3)
VHb
VHb-Red
Controlb>
Phosphate Pathway
0.26 ±0.06
0.35 ±00.6
0.46 ±0.05
0.68 ±0.07
0.46 ±0.05
R5P From GAP and S7P
0.84 ±0.02
0.81 ±0.02
0.92 ±0.02
0.86 ± 0.02
0.85 ±0.02
R5PfromG6P
0.16 ± 0.02
0.18 ±0.02
0.08 ±0.02
0.14 ±0.02
0.14 ±0.02
TCA
cycle
OAA from PEP
0.53 ±0.13
0.88 ±0.02
0.99 ±0.02
1.01 ±0.02
0.99 ±0.02
PEP from OAA
0.02 ±0.05
0.03 ±0.14
n.d.d>
n.d.d>
n.d.d>
Pyr
from malate
(ub)c)
0.00 ±0.04
0.00 ±0.16
n.d.d)
n.d.d)
n.d.d)
Pyr
from malate
(lb)c>
0.00 ±0.02
0.00 ±0.01
n.d.d)
n.d.d)
n.d.d)
0.01 ± 0.06
0.06 ±0.03
0.00 ±0.02
0.00 ±0.03
0.01 ± 0.02
0.70 ±0.09
0.60 ±0.11
0.19 ±0.06
0.20 ±0.09
0.25 ±0.04
om±om
0.19±0.04
0.65 ±0.03
0.73 ±0.04
0.71 ± 0.03
0.91 ± 0.03
0.88 ±0.04
0.87 ±0.03
0.86 ±0.04
0.96 ±0.03
ICT from Sue and GOX
(Glyoxylate shunt)
°^AJ!!?SiblyC0nVertedt0FUM
and SUCC
PYR
reversibly
converted to
AcCoA and FOR
AcCoA from PYR
CI metabolism
Serine from
Glycine
a)
Glycine
and CI
from CO2 and CI
Analysis
of the
0.22 ±0.04
0.24 ±0.03
0.22 ±0.02
0.16 ± 0.02
0.35 ±0.02
0.17 ±0.03
0.06 ±0.03
0.04 ±0.03
0.05 ±0.03
0.00 ±0.02
13C-labeling patterns of the proteinogenic
through a particular pathway of
fraction of carbon directed
values and standard deviations
b>
data
as
previously published
c) ub:
upper
boundary;
are
listed.
for E. coli
lb: lower
amino acids allowed
MG1655:pUC19 (Fiaux
boundary
d> not determined
88
calculating
the
the central carbon metabolism. Mean
et al.
1999)
Chapter
A)
3: Metabolism of
hypoxic hemoglobin-expressing
E. coli
Glucose
D-lactate
Formate
Ethanol
Acetate
sue
^k
OGA-DH
.
GOX
shunt
\
%
OGA
Formate
CIT
Ethanol
0.88
AcCoA
0.90
Acetate
glycolysis and the tricarboxylic acid cycle in the central
carbon metabolism of hemoglobin expressing microaerobic E. coli MG1655 cells. A) Flux ratios of
wild-type E. coli (Fiaux et al., 1999), VHb-expressing cells and VHb-Red-expressing cells (from top to
bottom of the boxes). B) Flux ratios of FHPg- and FHP-expressing E. coli (from top to bottom of the
boxes). Glycolysis (rectangular shape on the left) and the tricarboxylic acid cycle (circular shape on
the right) are highlighted. Enzymatically measured by-products and glucose are given in bold italics.
Carbon fragment patterns of the boxed metabolites were directly determined by [13C, aH]-COSY of
proteinogenic amino acids. The fractions of molecules given in square boxes are synthesized via the
reactions pointing at them, and numbers in ellipses indicate the amount of reversible
interconversion of the molecules. Enzyme names are given in italics. Active pathways are shown
with solid arrows. Dashed arrows indicate that the represented enzymatic conversion is inactive
(ME, GOX-shunt and OGA-DH). Abbreviations: AcCoA, Acetyl-coenzyme A; CIT, citrate; MAL,
malate; OAA, oxaloacetate; OGA, oxoglutarate; PEP, phosphoenolpyruvate; PYR, pyruvate; ME,
malic enzyme, PFL, pyruvate-formate lyase; PEP-CARB, phosphoenolpyruvate carboxylase, CITSYN, citrate synthase, GOX shunt, glyoxylate shunt; OGA-DH, oxoglutarate dehydrogenase.
Figure
3. Flux ratios and interaction of
89
Chapter
3: Metabolism of
An additional notable observation is that the
unlabeled molecules, which
would expect if
Table
4).
pyruvate
The 13C-enrichments of AcCoA and
therefore not
small that
strictly uniform,
they
were
ratios in the TCA
cycle
so
affected
by
decreased
that
such
reversibility
the
Finally,
we
by
based
tracing
on
configuration:
fragments
of the reactions
Sauer
et
connecting
(19, 27). Furthermore,
strains
activities could not be assessed
when the TCA
are
are
the earlier
pool supports
al.
E. coli cells
hypoxic
the
(41) postulated
AcCoA to acetate
glyoxylate
(Table 4),
suggestion
might
pools
and
well
of
as
presently
observed in FHP-
VHb-Red cells these
fragment,
since the Ca-C
as
a
pyruvate catalyzed
labeling pattern
VHb-
be
presence
shunt is not active in the
For
a
(Table 4).
the conversion of malate to
(Figure 3).
hardly
case
present observation of
which would be
traced from oxaloacetate to pyruvate to determine malic enzyme
generated
synthesis (see
is in any
fluxes between intra- and extracellular acetate
FHPg-expressing
one
intermediates
corresponding
intact C3
the malic enzyme does not contribute to the
and
the
detectable with the program FCAL; calculation of flux
also observed that the
studied cells
pool;
from those that
for AcCoA
source
of intra- and extracellular acetate in
exchange
diluted with
was
but the deviations from uniform 13C-enrichment
enrichment of the AcCoA
metabolic
reversible
not
pool
slightly
dilution at the level of AcCoA. The
a
isotope
exchange
general
AcCoA differed
the sole carbon
were
AcCoA
from the external acetate
probably originated
isotopomer abundances found for
13C
E. coli
hypoxic hemoglobin-expressing
activity,
is not
cycle operates in a branched fashion.
Discussion
The
expression
of VHb, VHb-Red,
FHPg
of
improve hypoxic growth properties
cultivations
positively
(12). Although
the
expression
modulate the microaerobic
underlying changes
of metabolic
and FHP
coli
E.
of these
growth
we
have
90
previously
MG1655
cells
in
hemoglobin proteins
shown to
bioreactor
is able to
of E. coli, little is known about the
configurations
heterologous globins. Therefore,
was
induced
studied
by
the
expression
of these
microaerobic
carbon
the
3: Metabolism of
Chapter
13C-labeling
by-product
the
more
(43-46).
and 2D NMR spectroscopy
accurately
The
one
hand and
the
changes
FHPg-
genetic approach
expression
has been
and
FHP-expressing
extensively
on
major
E. coli
VHb-Red-producing
E. coli strains
indirectly suggested
network to
results of Tsai et al.
1.5-fold increase in
controls. In addition, the
VHb-negative
coli, albeit
that
supported by
VHb-
by
mathematical model
different extents
to
more
aerobic
(48) revealing
a
d content in
cytochrome
specific activity
controls. The
of
regime.
This
hypothesis
5-fold increase in
o
o was
complex
was
cytochrome
as a
enhanced
by
50%
is able to extrude
proton pump (38). VHb-expressing cells do actually display
transmembrane proton
gradient
and
a
two
are
in
qualitative agreement
VHb-expressing
cells have
reentry of the proton flow
activity
and ATP
Keeping
production
VHb-expressing
integrate
expressing
E.
higher
findings
E.
coli.
of the
can
TCA
taking
an
cycle
These
that
showing
ATPase
into account the
present
interrupted
is
TCA
in
a
cycle,
growth
we
of VHb-
normally regarded
it has also been shown for
operate
91
(20, 48).
higher
up to 30%
model for microaerobic
growth. However,
cycle
controls
(8,20).
coli, which showed
Interruption
E. coli that the TCA
yields
in mind and
a
higher
ATP turnover rate than controls. The
relative to controls
all the information into
characteristic for anaerobic
growing
65%
to
31P NMR results
previous
into the cell via ATPase
the above-mentioned
data of the
tried to
a
with
a
not
of membrane-translocated
higher yield
protons per oxygen molecule reduced, when compared
findings
o
cells relative to
VHb-expressing
cytochrome
cytochrome
global
is able to shift the
VHb-expression
a
(20, 49). Thus,
protons per oxygen molecule reduced, whereas the cytochrome d complex does
function
on
the other hand.
Previously, a
studied in E. coli.
regulatory
relative to
analyzed
we
alleviate adverse effects of oxygen limitation
to
microaerobic
a
using
of carbon metabolism. Our results revealed
activate both terminal oxidases in E.
and
addition,
FHP
that VHb is able to increase the intracellular oxygen tension and to
conjectured
the model
In
or
excretion and intracellular metabolite content in order to unravel
differences in the metabolic states between VHb- and
the
E. coli
expressing VHb, VHb-Red, FHPg
metabolism of E. coli MG1655 cells
fractional
hypoxic hemoglobin-expressing
as
a
aerobically
branched fashion
once
the
Chapter
3: Metabolism of
cellular energy demands
of Tsai et al.
study
of
increasing
via AcCoA but
cycle
showed that the carbon fluxes
slightly
connecting glycolysis
cycle
interrupted
for VHb- and
to
increasing
above
a
to TCA.
Therefore,
generation
of
expressing
cells may then
data
on
on
reducing equivalents
satisfy
NAD(P)H
included
cells with the
similar flux ratios. However, additional data
formate and
to the
an
same
increased
hemoglobin expressing
hypothesis
that VHb- and
metabolism
more
these strains
of
consumption
strain
might
VHb-Red-expression may
not result from
cellular energy demands
These
can
a
leads
activity
to
a
decreased
in VHb-
and
growth
(11). Comparison
VHb-negative
glucose but a
(Table 1).
efficiently. Therefore,
TCA
study
previously published
some
conditions reveal
experimental
that the TCA
demands for
wild-type
on
the TCA
shown in this
cyclic
control cells in Table 4
VHb-Red-expressing
as
are
anaplerotic
the
Higher glycolytic activity
and ATP.
we
entering
hypothesize
may
cycle
A
consequence, the
concomitantly
and
sources
the ATP and
hemoglobin-negative
grown with the
we
coli. The absence of
support this hypothesis
VHb- and
a
through
threshold VHb concentration,
less efficient oxidation of carbon
NMR data
VHb concentration. As
increases the carbon flux
VHb-Red-expressing E.
maintenance. To
the TCA
through
amounts of VHb reduces the carbon flux
reaction
is
glycolysis (1).
satisfied with energy derived from
are
decreased with
concomitantly
expression
(49)
E. coli
hypoxic hemoglobin-expressing
of the
control reveals
£. coli MG1655 cells
of
higher production
smaller
growth rate
relative
may confirm
findings
our
enable the cells to operate their
the lack of functional TCA
cycle activity
in
anaerobiosis, but simply from the fact the
be satisfied
by
a
more
efficient ATP
generation
system.
Unfortunately,
in other
the
physiological
role of FHP either in its native host, R.
heterologous expression systems
is
poorly
characterized. FHP
eutropha,
or
expression,
like VHb, is induced under microaerobic conditions, and may interact with the gas
metabolism
during
denitrification in R.
not show any difference relative to
nitrite
as a
eutropha (9, 37). However, flip
wild-type
sole electron acceptor, but did not
Preliminary attempts
cells when grown
transiently
anaerobically
with
accumulate nitrous oxide.
to demonstrate nitric oxide reduction with
92
mutants did
purified
FHP and
Chapter 3:
NADH
as
Since
our
hypoxic hemoglobin-expressing
E. coli
unsuccessful
and
role
of
in the detoxification of nitric oxide is still under discussion
(24,
electron
an
flavohemoglobins
31).
Metabolism of
donor
were
results have shown that FHP
previous
E. coli under microaerobic conditions
able
hypothesis
to
a
higher
highly
is
free
the
increase
to
supported by
oxygen
(12),
intracellular
our
data
02-sensitive enzyme Pfl, which
in VHb- and
Recently,
VHb-Red-expressing
Gardner et al.
dissociation constant differs
uM-V1, koff
=
5600
(kon
to
that FHP- and
VHb, (K
k0ff 0.2
50 uM-V1,
to
FHPg-expression
leads
and inactivation of the
from the values
The
found to be active
=
0.014
oxygen
pM_1).
globin
on
to FHP
to
(K
=
for
that the
(kon
=
constants
high
Due to the very
respiratory complexes
mechanism of biochemical action of FHP
for VHb
equilibrium
affinity
properties
and found
reported
association
high
s-1),
koff, VHb-expressing cells have previously been proposed
possibly provide O2 rapidly
This
unexpectedly,
rather
was,
coli.
E.
in
FHP and determined the kinetic
strongly
s-1) (34, 51).
affinity
support growth of
concentration
oxygen
VHb-expressing cells
determined for FHP and VHb attribute
and low oxygen
to
cells.
(13) purified
and release
binding
oxygen
help
the
it would nonetheless appear that FHP is
showing
rate than in
uptake
can
(9),
250
78
(K)
pM"1)
values for
scavenge oxygen and
in E. coli
(20, 47-49).
The
the cellular metabolism of E. coli
is still unknown.
In this
study
improve
globin
reduce
we
have shown that inverse metabolic
metabolic
genes.
efficiency
of microaerobic cells
Through introducing FHP
and
FHPg
glucose consumption by approximately
affecting
cell
engineering (3)
by expression
into E. coli cells
can
of
or
able to
VHb-Red-
implications
for
expressing
cells without
economical
aspects of potential biotechnological applications, promising lowered
production costs.
93
This has direct
heterologous
we were
50% relative to VHb-
growth.
be used to
Chapter
3: Metabolism of
hypoxic hemoglobin-expressing
E. coli
Acknowledgments
This work
was
supported by
the ETH Zürich and the Swiss
for
Priority Program
Biotechnology (SPP2).
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Szyperski T., R. W. Glauser, M. Hochuli, J. Fiaux, U. Sauer, J. E. Bailey, and K. Wüthrich.
1999. Bioreaction network topology and metabolic flux ratio analysis by biosynthetic
fractional 13C labeling and two-dimensional NMR spectroscopy. Metabol. Engin. 1:189-197.
Tsai, P. S., G. Rao, and J. E. Bailey. 1995. Improvement of Escherichia coli microaerobic
oxygen metabolism by Vitreoscilla hemoglobin: new insights from NAD(P)H fluorescence
and culture redox potential. Biotechnol. Bioeng. 47:347-354.
Tsai, P. S., M. Nägeli, and J. E. Bailey. 1996. Intracellular expression of Vitreoscilla
hemoglobin modifies microaerobic Escherichia coli metabolism through elevated
concentration and specific activity of cytochrome o. Biotechnol. Bioeng. 49:151-160.
Tsai, P. S., V. Hatzimanikatis, and J. E. Bailey. 1996. Effect of Vitreoscilla hemoglobin
dosage on microaerobic Escherichia coli carbon and energy metabolism. Biotechnol. Bioeng.
49:139-150.
50.
Tyree, B., and D. A. Webster. 1987. The binding of cyanide and carbon monoxide to
cytochrome o reductase associated with cytochrome o purified from Vitreoscilla. J. Biol.
Chem. 249:4257-4260.
51.
Webster,
D. A. 1987. Structure and function of bacterial
52.
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proteins, p.
Inorganic Biochemistry, Elsevier, N. Y.
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245-265. In Advances in
96
hemoglobin
Chapter 4:
Bacterial
Evidence for role of bacterial
hemoglobins
and
hemoglobins
in NO* detoxification
flavohemoglobins for
of nitrosative stress in Escherichia colt
alleviation
Chapter 4:
Evidence for role of bacterial
hemoglobins
in NO* detoxification
Abstract
Escherichia
coli
MG1655
flavohemoglobin genes
cells
from
a
novel
expressing
medium copy number
bacterial
plasmid
were
flask cultures under nitrosative and oxidative stress. E. coli cells
proteins display
(SNP) compared
bacterial
of
Vitreoscilla sp.
has
been
enhanced resistance
to the control strain
(VHb)
specific
that
NO*
heterologous
in vitro NO*
a
from
detoxification
from
consumption
extracts from the
expressing
The
expression
and
Campylobacter jejuni (CHb)
is
different
challenged
also
a
taxonomic
cells. In addition, it
groups
of
feature
common
and
can
be
wildtype
a
assay. Protein extracts isolated from E. coli strains
more
readily
consumed
strain. An oxidative
authentic
challenge
to the cells evoked
Improved
had also been observed when the
NO" than
oxidative stress-
flavohemoglobins
from E.
coli, Klebsiella pneumoniae, Deinococcus radiodurans and Pseudomonas aeruginosa
expressed
these
sodium-nitroprusside
parental plasmid.
non-uniform response of the various cell cultures.
sustaining properties
grown in shake
host. These observations have been confirmed in
overexpressing flavohemoglobins
protein
the
conferred resistance to SNP
flavohemoglobins originating
a
the NO* releaser
bearing
hemoglobins originating
shown
transferred to
against
and
hemoglobin
in E. coli.
98
were
Chapter
4: Evidence for role of bacterial
hemoglobins
in NO* detoxification
Introduction
Oxidative and nitrosative stresses
and
species (ROS)
oxygen
nitrogen species (RNS)
and noxious metabolic side
such stresses
of aerobic
lifestyle.
ROS
are
(NO*) (nitrogen
either formed
are
Oxidative stress is
formed in vivo
(19,52).
living organisms.
In
an
reduced
when molecular
flavoproteins
(25, 32). RNS,
such
monoxide is the IUPAC recommended
potent peroxynitrite (ONOO-)
directly
cause
indirectly
or
undesired
as
unavoidable side effect
during respiration
particular
Reactive
challenged by
also be
organisms might
or
in this redox process in Escherichia coli
oxide radical
more
products
generated exogenously.
oxygen is reduced to water
implicated
encountered in all
are
have been
the nitric
as
name)
and the
nitrosative stress
(27).
In mammals elevated levels of NO* in combination with ROS
in cellular responses
plants,
against infections, foreign
play key
ROS appear to
pathogens.
ROS
seem
presence of NO" in
to act both
plants
that NO* appears to
early
roles in
as
cellular
bodies and
and late
neoplastic
roles
during
the
In
denitrifying bacteria,
used
as
NO* is
plant-pathogen
as
E.
and
membranes
can
nitrosative
occur
of the cell's
(constitutive
and
by
an
damage
to
proteins,
for elimination
inducible) enzymatic
under the control of the soxRS and
(4).
part of host
as
in
nitrite
or
are
non-denitrifying
reactions
(56)
or
by
(5).
nucleic
when the concentration of reactive
mechanisms
interaction
when nitrate
(14, 20). Additionally,
oxidase
The
plants.
coli, NO" may be produced by non-enzymatic
reduction of nitrite to NO*
Oxidative
and
produced during growth
terminal electron acceptors
bacteria such
capacity
invading mammals
(12).
to
suggest clearly
Therefore, infectious bacteria may be challenged by ROS and RNS
defense mechanisms, when
toxins
tissues. In
molecules
killing
is also well documented and recent results
play important
potent
stages of plant response
and
signals
serve as
(16).
mechanisms to
oxyR regulatory
99
E.
acids
species
cellular
exceeds the
coli has
detoxify
networks
and
developed
ROS and RNS
(9,13, 30,46).
Chapter
4: Evidence for role of bacterial
Whereas the induction of
of E. coli
was
hmp expression by
(21, 28).
HMP
plays
in E. coli under aerobic conditions
the
heme-bound
considered
to
be
been
proposed (29),
The
impact
main
physiologically
at
the accumulation of
also
more
flavohemoglobins
or
Salmonella
such
sp.
as
(35).
of
a
heterologous
The
an
dioxygenase (21, 28)
nitrate, has been
Alternatively
degradation.
NO*/02
for
a
concentrations has
has
organisms
or
improved growth
a
subject
of
of native HMP leads to
E. coli
an
hmp
null
(43).
oxidative stress response have
flavohemoglobins
from
diverse
tuberculosis
(55), Mycobacterium
(31),
eutropha (22).
single
express
some
domain
hemoglobin
bacterial
yields
the
ROS
is
stress
In contrast,
flavohemoglobins,
homodimeric
VHb
oxidative
against
cerevisiae
express
protein
relevant
on
hemoglobin.
is the
bacteria such
as
hemoglobins (53, 3).
one
from Vitreoscilla
It has been
postulated
that
increased intracellular oxygen concentration
activities
been
such
are
stimulated in recombinant host
successfully
as
E.
(CHO)
and enhanced
100
expressed
in
various
coli, Bacillus subtilis, Streptomyces
S. cerevisiae and Chinese hamster ovary
VHb results in
a
yield
overexpression
and Ralstonia
dependent cellular
VHb
biotechnologically
coelicolor,
species
VHb is
and in turn oxygen
cells
Saccharomyces
thoroughly investigated
expression
sensitive
Campylobacter jejuni
(VHb) (53, 39).
to
relevant
roles in nitrosative
typhimurium (10,11),
Vitreoscilla sp. and
including
peroxide (1, 44).
demonstrated
or
While most bacterial
The most
more
putative
proposed
microorganisms
and
superoxide
has been found to be
been
the
where heme-bound NO" reacts with oxygen to form nitrate.
of bacterial
Similar functions
catalyzed by
Although
of O2 and NADH.
NO*
discussion. It has been shown in E. coli that
mutant
nitrosative stress
against
The reaction
48).
NO"
with
for
route
implication
two different mechanisms for the HMP
A route
reacts
the
(47),
role in the detoxification of RNS
review:
a
consumption
proposed.
oxygen
the
'denitrosylase' activity
for
widely accepted,
mediated reaction have been
where
known
was
significant
a
(43,
HMP converts NO* into nitrate with
overall reaction balance is
NO*
in NO* detoxification
HMP in the inducible response
flavohemoprotein
derived later
hemoglobins
cells. The
productivity
expression
of
of recombinant
Chapter
proteins
or
4: Evidence for role of bacterial
improved synthesis
37). Recently,
similar
of
primary
growth-stimulating
have also been observed when novel
from several bacterial
In this
study,
aeruginosa,
R.
species,
have
we
Salmonella
the
Klebsiella
of these
and
hemoglobins
oxidative
jejuni
to
effects and
expressed
pneumoniae,
bioremediation
(45),
view
the NO
of
to
carbon utilization
in E. coli
(3,17).
genes from Pseudomonas
goal
hemoglobin
to
was
heterologous
study
genes from
the
feasibility
host from nitrosative
like
biotechnological applications
further
consumption
review:
which have been isolated
and the
a
(for
Deinococcus radiodurans, E. coli and
in E. coli MG1655. Our
In
stress.
improved
flavohemoglobin
provide protection
metabolites
secondary
or
eutropha (previously Alcaligenes eutrophus)
Vitreoscilla sp. and C.
in NO* detoxification
flavohemoglobins,
have been
expressed
typhi,
hemoglobins
activities of these
hemoglobins
have also
been tested in vitro.
Materials and Methods
Bacterial strain,
plasmids,
E. coli MG1655 K-12
throughout
this
cultivations
were
4
g/1 glucose,
vitamin mix
(F
,
study.
and
growth
À ; Cold
Spring Harbor Laboratory)
Plasmids used in this
performed
or
study
MgSO-i,
with
elements
(36),
(LB, 50). Ampicillin (100 mg/1)
was
(5 mg/1),
trace
maintenance. To obtain identical cultivation
plasmid
conditions for
and for oxidative and nitrosative stress
shake flasks
flask
capacity.
rpm. Cell
with
were
a
filled with media to
All cultivations
growth
was
Perkin Elmer XI
followed
a
fixed ratio of
1/10
performed
at 37°C
by measuring
culture
were
All
listed in Table I.
added to all cultures for
precultures
host
as a
(50) supplemented
thiamine
in Luria-Bertani medium
has been used
are
either in M9 minimal medium
0.1 mM CaCb, 1 mM
(36)
conditions
spectrophotometer.
101
experiments,
of medium to total shake
on a
rotatory shaker
turbidity
at 600
nm
at 300
(Aöoo)
Chapter 4:
Evidence for role of bacterial
Table 1. Plasmids used in this
Flavohemoglobin/
Plasmid
Origin
Hemoglobin*
"
in NO* detoxification
hemoglobins
of
study.
Reference
protein
(36)
pKQV4
None
pPPCl
VHb*
pAXl
FHPg*
R.
pAX4
VHb-RedD
Chimeric
pAX5
FHP
R.
pECS14
HMP
E. coli
(3)
pBSS15
HmpBs
B. subtilis
(3)
pPAS6
HmpPa
pDRS3
HmpDr
pCJS8
CHb*
pSTS28
HmpSt
pKPSlO
HmpKp
Fusion
protein
(36)
Vitreoscilla sp.
P.
(17)
eutropha
(17)
protein
(17)
eutropha
(3)
aeruginosa
(3)
D. radiodurans
jejuni
(3)
typhimurium
(3)
pneumoniae
(3)
C.
S.
K.
of Vitreoscilla VHb and the C-terminal reductase domain of R.
eutropha
flavohemoglobin FHP.
Determination of resistance to SNP, and
Precultures
were
paraquat
inoculated to Aôoo of 0.1 from
various bacterial strains. At Aöoo of 0.5 the
obtain
a
1. To determine the resistance of
strains
precultures
final concentration of 0.1 mM and the
and control strain
precultures were
toxic
were
ROS and
nitroprusside-sodium-dihydrate (SNP, Fluka),
initial Aöoo of 0.2. As
a
dichloride
reference,
under identical conditions and Aöoo
an
RNS
species,
1 mM
free cultures
either 1 mM
containing
K4Fe(CN)6
were
also
and stressor-free cultures
was
routinely
Aéoo of
IPTG induced
or
100 uM
(paraquat, PQ, Fluka), respectively,
stressor
preculture. Both, stressor-containing
grown to
flavohemoglobin-expressing
and
used to inoculate fresh minimal medium
dimethy1-4,4'-bipyridinium
induced with IPTG to
precultures were
hemoglobin-
against
grown cultures of the
overnight
measured
prepared
were
during
1,1'-
to
an
from each
grown further
a
three hours
period.
Specific growth
the natural
rates
(p)
logarithms
were
calculated
of Aöoo values
by applying
linear
plotted against
102
regression analysis
the time
course
on
of the
Chapter
4: Evidence for role of bacterial
cultivations. The ratio of p from
a
reference culture
from the
respective
quantify
the effects of the stressors
performed
and
ANOVA
and
triplicate
in
culture
Dunnett's
subjected
(pno stress)
on
repeated
hemoglobins
growth
in NO* detoxification
to
stressor
a
(pstressor /
calculated
was
(pstressor)
pno
to that
stress)
of the E. coli cultures. The assay
test
for
applied
was
was
experiment. One-way
at least twice for each
multiple comparison
to
statistical
analysis.
Growth of cells and
preparation
of soluble
protein
fraction for in vitro NO*-assay
ampicillin (100 pg/ml).
E. coli cells
were
grown in LB medium in the presence of
Precultures
were
diluted into fresh LB medium to Aôoo of 0.1 and grown to Aôoo of 1
where the
of the
expression
the addition of IPTG to
10 hours at
a
low
a
hemoglobins
and
flavohemoglobins
was
final concentration of 1 mM. The cultures
shaking
rate
of 170 rpm to
were
production
ensure
induced with
grown for
of the heme
cofactor.
Cells
were
by centrifugation
harvested
and
resuspended
in assay buffer
(50
NaCl, 1 mM EDTA, 100 mM Tris-HCl, pH 7). Soluble protein fractions
obtained
as
described
Determination of
Total soluble
method
were
(17).
protein
protein
mM
and heme concentration
concentration of the extracts
(6) using reagents
was
determined
obtained from BioRad and bovine
by
serum
the Bradford
albumin
as a
standard.
Heme content in crude extracts
method
40%
(2). Briefly,
pyridine
an
equal
and 0.8 mM
was
determined
volume of
K3Fe(CN)6
a
was
stock solution
added to
fraction and mixed. Absorbance of this oxidized
and
539
nm.
A
few
grains
using
an
the
containing
aliquot
sample
of sodium dithionite
pyridine
was
were
hemochrome
200 mM NaOH,
of the soluble
protein
measured at 556
added
to
nm
reduce the
hemichromes to hemochromes and the maximum and minimum absorbance values
103
Chapter
(556
nm
4: Evidence for role of bacterial
and 539 nm,
respectively)
hemoglobins
in NO* detoxification
measured after 3 min of incubation at
were
temperature. The absorbance values of the oxidized form
values recorded for the reduced
subtracted from the maximum absorbance. A
generated using
was
mM-1 cm-1
hemin
(Sigma)
as a
obtained for hemin and
was
the
and
sample,
pyridine
were
room
subtracted from the
absorbance
minimum
hemochrome standard
was
curve
standard. An extinction coefficient of 19
was
used to calculate heme contents of the
samples.
Measurements of nitric oxide
Gaseous nitric oxide
passed through
pellets
to
by degassing
(Höllriegelskreuth, Germany)
obtained from Linde
degassed
a
remove
was
consumption
NaOH solution and
higher nitrogen
oxides before
a
use.
column
packed
NO* solutions
assay buffer for at least 30 min with N2 and then
and
with NaOH
prepared
were
saturating
it with
NO".
NO'
decay
was
measured with
an
ANTEK Instruments nitric oxide
chemiluminiscence detector, which
ml stirred cuvette connected to
of NO*
An
corresponds
appropriate
to
a
signal
a
was
adapted
to
liquid
capillaries
bundle of
measurements
solution
was
as
amount of crude extract
indicated in the results section
as
baseline and after 10 seconds NO"
(2 mM) using
gastight SampleLock
recorded until the baseline
room
temperature and
crude extracts
and
a
expressed
were
in
was
was
added from
Hamilton
NO
syringe.
consumption
by subtracting
a
2
relative to heme content.
104
was
added to
a
of this mixture
an
NO* saturated
The
were
rates in
the rate of NO*
was
signal
reached. All measurements
duplicates.
calculated
using
a
of 42 mV.
final concentration of 200 uM unless otherwise stated. The
set
with
that allow gas diffusion. luM
added to the cuvette and filled to 2 ml with assay buffer. NADH
was
analyzer
decay
of NO*
performed
at
the presence of
consumption
in buffer
Chapter
4: Evidence for role of bacterial
hemoglobins
in NO* detoxification
Quantification of nitrate and nitrite
Nitrate and nitrite concentration in
Nitrate
reagent.
enzymatically
was
colorimetric assay kit
directly
(Roche
were
analyzed by using
was
converted
to
and
nitrite
the Griess
quantified using
was
determined
(Molecular Probes, Eugene, Oregon).
Nitrate and
Molecular
with the Griess reagent
nitrite standards
samples
Biochemicals).
used for calibration. Each
sample
Nitrite
was
measured twice.
Results
Determination of resistance of
coli cells
against
Table 2. Ratios
SNP and
pstressor/ uno stress
hemoglobin
and
flavohemoglobin expressing E.
paraquat
of cultures
exposed
to SNP
or
paraquat
and of stressor free cultures
Strain*)
1 mM SNPb>
Control
0.68 ± 0.06c>
FHP
0.88 ± 0.02
VHb-Red
0.88 ± 0.05
P
<
0.001
HmpKp
0.84 ± 0.03
P
<
0.001
HmpPa
0.86 ± 0.04
P
<
0.001
HmpSt
0.89 ± 0.05
P
<
0.001
HmpBs
0.92 ± 0.06
P
<
0.001
HMP
0.90 ± 0.08
P
<
0.001
HmpDr
0.93 ± 0.02
P <, 0.001
FHPg
0.92 ± 0.02
P
<
0.001
VHb
0.91 ± 0.06
P
<
0.001
0.87
P< 0.001
CHb
+
P
0.07
0.001d>
<
~
HMP«)
0.99 ± 0.08
P
<
0.001
VHbe>
0.96 ± 0.06
P
<
0.001
hemoglobins and flavohemoglobins in E. coli MG1655 cells. The control
parental plasmid pKQV4.
b) Ratio of
growth rates from a culture exposed to SNP and a stressor free control culture.
c> Mean values and standard deviation are
given. Values are calculated from the results of at least
two independent experiments performed in triplicates.
d) Statistical
significance data has been analyzed using ANOVA and compared using Dunnett's
multiple comparison test assuming a confidence interval of 99%. All data has been compared to the
a>
Strains
expressing
various
strain contained the
control cultures.
e)
Cultures of E. coli
MG1655:pPPCl
and
MG1655:pECS14
105
grown in the presence of 1 mM
K4(Fe(CN)6).
4: Evidence for role of bacterial
Chapter
E.
coli
cells
expressing
significantly higher
control strain
/pno
of
either
expressing
coli cells
All recombinant E. coli strains
or
responsible
either type of
for
performed.
Growth rate of
VHb
or
FHP
not affected
were
expression
SNP.
are
of these
that
the presence of this
affected
possible
flavohemoglobins.
of
cyanide
IQ(Fe(CN)6) containing
by
and
cultures
by
hemoproteins helps
However it is not
equally capable
eventuality
coli
E.
only minimally
by hemoglobins
proteins
of
show psNP
protecting the
to
£.
cells
liberated from SNP
1 mM
K4(Fe(CN)6)
cultures
expressing
complex
have
either
relative to control
(Table 2).
Table 3. Ratios
pstressor/ uno stress
of cultures
Strain")
a) Strains
by
are
investigated
rates
growth inhibition, experiments using
been
cultures
the
exerted
from nitrosative stress. To exclude the
is
hemoglobins
between the effects exerted
expressing
Thus, growth
0.9.
clearly indicating that
toxicity
achieved
hemoglobins
or
ratios relative to the ratio obtained with the
stress
approximately
the cells to resist the
in NO' detoxification
flavohemoglobins
flavohemoglobins
nitrosative stress,
distinguish
/pno
(0.68) (Table 2).
ratios
stress
psNP
either
hemoglobins
exposed
to 100
pM paraquat and
of stressor free cultures
paraquatb>
100 uM
Control
0.32 ± 0.06c>
FHP
0.36 ± 0.04
HmpKp
0.33 ± 0.08
P S 0.05
HmpPa
0.43 ± 0.05
P
HmpSt
0.48 ± 0.05
HmpBs
0.24 ± 0.01
HMP
0.52 ± 0.07
P
<
0.001
HmpDr
0.52 ± 0.05
P
<
0.001
VHb
0.32 ± 0.06
P
>
0.05
CHb
0.28 ± 0.05
P
>
0.05
P
0.05d)
P
>
P
>
<
>
0.05
0.001
0.05
hemoglobins and flavohemoglobins in E. coli MG1655. The cells, control
parental plasmid pKQV4.
growth rates from a culture exposed to paraquat and a stressor free control culture.
expressing
various
strain contained the
b)
Ratio of
c>
Mean values and standard deviation
given, standard deviation is calculated from the results of
independent experiments performed in triplicates.
d> Statistical
significance, data has been analyzed using ANNOVA and compared using Dunnett's
multiple comparison test assuming a confidences interval of 95%. All data has been compared to the
respective control cultures.
are
at least two
106
Chapter 4:
The effect of the
Evidence for role of bacterial
paraquat
Increased resistance
MG1655
HmpPa.
strains
the
expressing
maximally by
Pparaquat/pno
more
the
NO*
were
of
an
decay
rates in
the
by
a
consumption
accelerated
of
a
CHb
compared
paraquat
were
resulting
in
expressing
significantly
differ
(0.32) (Table 3).
assay
by flavohemoglobins
protein-free
to
and
treatment and the ratios
analyzer.
was
Soluble
a
fractions
reducing agent,
hemoglobins
and
protein
started with the addition
NO* saturated solution. In the absence of
of NO*
in the
seen
VHb
or
value which does not
added to the reaction chamber and the reaction
aliquot
only
was
the presence of
paraquat
followed with the nitric oxide
was
paraquat
HmpBs, HmpKp,
affected
from the ratio of the control strain
consumption
from
heterogeneous.
relative to the stressor free culture
50%
Pparaquat/pno stress were approximately 0.3,
In vitro NO*
of the cultures is
growth
specific growth
severely
in NO* detoxification
flavohemoglobins HMP, HmpSt, HmpDr
ratios of about 0.5.
stress
were
on
against O2"- generated
In these cultures the
decreased
cultures
treatment
hemoglobins
buffer. Therefore, NADH
the
was
only slightly
was
added to the
reaction mixture.
The addition of NADH
the
decay
observed in
of NADH
was
dramatically
protein-free solutions
necessary for both
hemoglobins,
such
concentration
as
as
CHb
turnover of the different
(Table 4).
were
not
in the presence of NADH. The addition
such
as
FHP, and classical
The control cell extract, at the
CHb and FHP, did not show any acceleration in NO*
consumption activity
subtracting
of NO* relative to
consumption
flavohemoglobins,
(Figure 1).
presence of additional NADH
NO"
increased the
rate of NO
The NO*
(data
flavohemoglobins
decay
heme
decay
in the
shown).
of various cell extracts is
and
in buffer and
consumption
significantly
not
same
rates for
displayed
hemoglobins
standardizing
in
was
Figure
2. NO*
calculated
by
to the heme content
VHb, CHb, HmpDr, FHPg and VHb-Red
different from the rate of the control. The
107
protein
extracts
Chapter 4:
containing
FHP and
NO* turnover of
containing
coli
Evidence for role of bacterial
the
showed
HmpPa
had
approximately
flavohemoglobins
highest
activities
numbers for the native
in NO* detoxification
hemoglobins
intermediary
leading
rates,
to
an
10-fold relative to controls. The
of K.
being
pneumoniae
S.
typhimurium,
100-fold
to
up
,
higher
acceleration of
protein
extracts
B. subtilis and E.
than the turnover
hemoglobins (Table 4).
®—
Buffer
a—
CHb
CHb
+
NADH
+
NADH
®—
Buffer
o—
FHP
%
500
FHP
+
+
NADH
NADH
>
G
40
20
60
80
Time
Figure
1.
NO"
hemoglobin
was
consumption
CHb and R.
measured with
performed
with
or
a
curves
eutropha
100
120
140
(s)
of soluble
protein
fraction of
cells
expressing
FHP in the presence and absence of NADH. NO"
nitric oxide
analyzer using
a
jejuni
consumption
chemiluminiscence detector. The assay
without the addition of 200 uM NADH. 42 mV
108
C.
corresponds
to 1 uM NO*.
was
Chapter
Due
to
the
fast
typhimurium,
K.
concentration
significantly
4: Evidence for role of bacterial
NO*
removal, the NO*
pneumoniae,
of 20
uM.
turnover
B. subtilis and E. coli
The
rates
at
a
in NO* detoxification
was
higher
a
of
extracts
also determined at
NO* concentration
S.
NO"
a
were
not
substantially higher
rate at 20
pM relative
to
the
one
pM (Table 4).
Table 4. NO'
consumption
of soluble
protein
fractions.
NO' turnover rates
protein
[s_1]
b)
extract11
10 uM NO'
a)
of cell
rate
different from the turnover determined at 10 uM besides for the extract
of B. subtilis, which reached
at 10
hemoglobins
20 uM NO'
d.c>
control
1.9 ± 0.1
control
2.0 ± 0.1
n.
d.
CHb
0.4 ± 0.1
n.
d.
FHPg
3.4 ±1.3
n.
d.
VHb
0.9 ± 0.1
n.
d.
VHb-Red
4.3 ±1.0
n.
d.
HmpDr
3.1 ±1.5
n.
d.
FHP
15 ± 1
n.
d.
HmpPa
26 ± 4
n.
d.
HmpKp
95 ± 1
86 ± 9
HmpSt
76 ± 4
78 ± 6
HmpBs
68 ± 11
128 ± 6
HMP
91 ± 4
90 ± 4
Soluble
protein fractions were obtained from
hemoglobins or flavohemoglobins.
b> NO' turnover rates were
calculated with
a
expressed
c> n.
d.: not determined. See text for details.
E.
coli
correction for
relative to heme content.
and
n.
109
MG1655
backgroud
cells
overexpressing
rates of NO'
either
decomposition
Evidence for role of bacterial
Chapter 4:
0
20
40
Figure
hemoglobin
0.05
consumption
and
an
consumption
corresponds
air
aliquot
was
buffer
equilibrated
of
a
(s)
protein fractions containing
Protein extracts
a
nitric oxide
stoichiometrically yields
prove the involvement of
a
nitrate
pM
saturated solution
flavohemoglobins
NADH have been
was
injected
minutes of incubation the
was
a
final concentration of
was
pM.
initiated
by
NO*
chemiluminiscence detector. 42 mV
(21, 23, 28).
aqueous-phase
used for determination of NO"
buffer and 200
NO"
a
final concentration of 10
analyzer using
removal, nitrate and nitrite concentrations
as
added to
it has been shown that the aerobic detoxification of NO*
reaction of NO* and Oi in the
such
were
various bacterial
200 uM NADH. The reaction
containing
NO" saturated solution to
followed with
120
100
to 1 uM NO".
Previously,
coli HMP
of soluble
flavohemoglobin proteins.
mg/ml in an
addition of
curves
in NO* detoxification
80
60
Time
2. NO"
hemoglobins
recovered
as
pM)(Table 5). Thus,
to
samples
nitrate, nitrite
and
were
determined. Identical mixtures,
in sealed
glass
air
equilibrated
vials and
pM.
for nitrite and nitrate.
almost undetectable in all
a
NO*
After 30
Mainly
all
samples (0-1
these data confirm that the acceleration of NO*
110
To further
in the observed NO'
final concentration of 20
analyzed
E.
nonenzymatic
(54).
nitrite
consumption, including
a
was
produce
hemoglobins
prepared
give
were
should
In contrast, the
by purified
decay by
Chapter 4:
hemoglobin-
and
presence of these
Evidence for role of bacterial
hemoglobins
flavohemoglobin- containing protein
proteins
and not to any
Table V. Nitrate
production
unspecific
of cell extracts
Protein extract3)
a>
Soluble
or
during
pKQV4
18 ±5
FHPg
19 ±5
VHb-Red
15 ±5
FHP
19 ±6
VHb
17 ±4
CHb
16 ±3
HMP
22 ±1
HmpSt
23 ±3
HmpKp
18 ±4
HmpBs
24 ±4
HmpPa
24 ±7
HmpDr
26 ±3
from
E.
coli
due
the
to
[uM]b>
Nitrate
obtained
is
NO" turnover.
17 ±4
were
extracts
reaction.
wt
protein fractions
hemoglobins
in NO* detoxification
cells
MG1655
overexpressing
either
flavohemoglobins.
Nitrate recovery after conversion of 20 uM NO" by cell extracts. Mean value of two
assays and standard deviation are given. Each measurement has been done twice.
b>
independent
Discussion
Several lines of evidence indicate that
analyzed
in this
response
against
study
are
either
hemoglobin
directly
or
and
indirectly
oxidative and nitrosative stress. In
have evaluated the effect of
flavohemoglobin proteins
involved with the cellular
our
hemoprotein expression
in vivo
on
experiments
the resistance
we
against
oxidative and nitrosative stress.
E. coli cells
expressing
the different
flavohemoglobins
uniform response to oxidative stress. When
environment,
growing
and
hemoglobins
cells in
a
statistically significant growth ameliorating
observed for E. coli cells
expressing HMP, HmpSt
111
and
had
a non¬
paraquat containing
effects
HmpDr.
were
only
The other strains,
Chapter
including
4: Evidence for role of bacterial
producing strains,
VHb and CHb
that observed for the control strain
the
Although
the
organisms
and oxidative stress
these
inhibitory
null mutant and
significantly
Therefore,
All
(43).
of
hmp
these
on
issues
point
the response
sustaining growth
of
and
the
with
experiments
superoxide (41). Anjum
and
plasmid-bearing
wild-
null mutant has been found to be
more
to
hemoglobins
experimental
strain
a
to the
might
exhibit
coli
regulatory
a
strain.
role rather
important role
an
capability
the
oxidative stress. This could be
tested in this
study
of
dose
one
of the
flavohemoglobins
were
host relative to the
of these
and
equally capable
non-expressing
pneumoniae,
fastest. This is not
surprising
S.
proteins
to
degrade NO',
of B. subtilis and of the
flavohemoglobins
of reactive
for
of
control
conditions.
bacteria E. coli, K.
amounts
a
HMP
against
heterologous
a
When tested in vitro the
containing
of E. coli HMP from
vitro
In
an
present study.
tested in the
strain under the
observed between
overproducing
paraquat relative
E. coli
were
(10). Overexpression
generation
various
differences in the
paraquat than the corresponding wildtype E.
flavohemoglobins
high
significant
no
paraquat and H2O2
strain
from
clear indication for the role of
for the differences observed between the different
hemoglobins
than
growth pattern
of the direct stress response in E. coli mechanism under oxidative
being part
reasons
typhimurium
In S.
sensitive to
an
same
flavohemoglobins
numerous, no
it has been concluded that HMP
flavohemoglobin
All
more
towards
sensitive
of
caused oxidative stress in vivo.
strain. In contrast,
stress
are
wildtype
showed the
confirmed these results and stated that
colleagues (1)
type
association
HMP also demonstrated the
purified
than
a
in NO* detoxification
(Table III).
concentrations of
multicopy plasmid
was
on
obvious.
is
proteins
minimal
hmp
reports
hemoglobins
and P.
typhimurium,
since
pathogenic
nitrogen species
when
the cell extracts
putative pathogenic
aeruginosa proved
bacteria
are
infecting
to be the
potentially exposed
their host
organisms.
to
It is
is up to the millimolar range,
reported
that the level of NO* in
although
the exact concentration of NO' in the bacteria is not known. Furthermore,
macrophages
112
Chapter 4:
Evidence for role of bacterial
in the denitrification process NO' is
M
(26).
NO' turnover of
of FHP and
are
in
NO'
purified
supposed
HMP
or
consumption
Vitreoscilla and C.
remediating
of HMP
as
is involved in the
the re-reduction of the
explanation
nitrate with the
by
our
by
any
HMP converts
consumption
would not be
like in
hemoglobins,
as
effective
in vivo.
as
as
O2
amounts of NO'and
O2
encountered in the in vivo assays.
equistochiometric
of NADH
hemoprotein-mediated
The NO" removal
(21, 23, 23, 28).
hemoproteins yields mainly
unspecific
membrane
contained the soluble
mainly
nitrate. Thus,
conclude that the observed acceleration of NO' removal
indeed due to the
a
is that the conditions of the in vitro assay, such
ones
is in
clear nitrosative stress
a
reduction of these
proteins
from
degrading activity
NO"
where
As the in vitro assay
and NO" concentration, differ from the
The NO' detoxification
significant
enzymatic
protein fraction,
Another feasible
a
(22).
shown. This could be due to the fact that
was
myoglobin (40).
by
well
as
hemoglobins
the
harboring
experiments,
the reduction of
caused
cell extracts
containing
numbers of Gardner et al.
The absence of
jejuni.
effect
protein
mediated
to attain concentrations of 10-9 -IO-7
small for cell extracts
was
contrast to the results of the in vivo
to
in NO' detoxification
yeast flavohemoglobin (22, 23, 28, 44) has been reported. Our numbers
good agreement with the reported
bound
hemoglobins
it is
plausible
by protein
extracts
to
is
conversion of NO' to nitrate and not
interaction of NO" with other
proteins contained
in the
cell extracts.
The results of the in vivo
CHb)
and
overcome
flavohemoglobins
are
the nitrosative stress
reductase domain
been included:
eutropha FHP,
consisting
experiments suggested
a
more
strain
termed
equally capable
produced by
carefully,
engineered
FHPg
and
the
to
a
that bacterial
of
supporting
SNP in vivo. To
following proteins
solely
express the
strain
hemoglobins (VHb,
producing
the E. coli cells to
study
the effect of the
described before have
hemoglobin
a
novel
domain of R.
fusion
protein
of N-terminal VHb fused to the C-terminal reductase domain of FHP,
termed VHb-Red
(17).
113
Evidence for role of bacterial
Chapter 4:
The results of the in vivo
conditions in respect to the
conditions. Both
and its fusion
when
the addition of the C-terminal reductase domain
or
significantly change
do not
pairs
of
the
protective
corresponding
native
and the
proteins (FHP
protein) perform equally
challenged
in NO' detoxification
that the removal of the C-terminal
experiments suggest,
reductase domain from FHP
(VHb-Red)
hemoglobins
effect under nitrosative stress
proteins
under
domain thereof
hemoglobin
well under
experimental
our
experimental
our
with SNP. However, the in vitro NO'
or
VHb
conditions
clearly improved
in the presence of the reductase domain. The inclusion of
reductase domain
improves
factor of 4-5.
explained by
necessary for the full
the
performance
of
FHPg
and VHb
a
hemoglobins by
a
this different behaviour between the effects exerted in vivo and
Again
in vitro could be
explain
the
is
consumption activity
the lack of
of
activity
an
appropriate external
hemoglobins
superior performance
of
in
respect
system
It would also
analysis.
in the in vitro
flavohemoglobins
reductase
hemoglobins
to
under those conditions, since the necessary re-reduction of the heme iron for the
NO' turnover is
bound to the
reductase
VHb
as
manner.
also
as
reductase
probably
domain. In
copurified
allows
previous studies
from Vitreoscilla
us
to
suggest that
as
active ferrous form. A
ferredoxin
a
recent
study,
NO*
Kaur et al.
previously
consumption activity
the VHb
VHb,
(53, 24, 33),
an
analogy
to
NADH-cytochrome
which
degradation by
an
was
variety
of
dependent
the addition of NADH
Vitreoscilla
possible
potentially
(38)
function
have also
described
analyzed
by Frey
in the extracts
protein. Unfortunately, they
an
endogenous
hemoglobins
reductase
systems
would have allowed the direct
et al.
comparison
114
to
exists
reductase
heme reductases.
the effect of addition of
by using
containing
do not
as
o
able to reduce
NADH
to reduce the heme-iron of
reductase domain to VHb in SNP stressed cells,
that of VHb-Red,
on
NAD(P)H oxidoreductase, NAD(P)H:flavin
and ferric reductase, which could
In
in
system of E. coli might be able
coli, such
effective if the reductase domain is
more
number of artificial electron acceptors in
a
biochemically
in E.
to be
The observed acceleration of NO*
(Figure 4)
the
globin
was
well
going
a
a
very similar construct to
(17). They
also showed
the chimeric
report any NO*
protein
turnover
of the data. Similar
as
faster
a
relative to
value, which
in this
study,
Evidence for role of bacterial
Chapter 4:
differences
more
recently
improve growth
and also
between
FHP and related
and recombinant
higher eukaryotes (37).
oxygen levels.
this leads to
Concomitantly,
increased levels of the
cytochromes,
cytochromes
of
or
ATP
and
VHb
FHP
is
endogenously produced
uptake.
of
In
VHb
strain
or
providing
(38)
higher
cytochromes
species
potential
to
the
speculate
uptake
same
than
(18).
E.
uptake
as
of the
respiratory
rates
a
FHP-or
coli
strains
These results
NO* turnover rate of
functions would
from
the intracellular oxygen
rates
effects
the
that the role
chain
respiratory
showed increased oxygen
higher
in
are
FHP/FHPg
cytochromes by
NO*
attributed to VHb in
require
a
an
close interaction of
preferentially
chain in both the native host
(49).
hemoglobins
have been
myoglobin
thereby protect
and indeed VHb has been shown to be
surrounding
Vitreoscilla and also in E. coli
bacterial
of
and
Prevention of the inactivation of
role. Both
novel
the inducible
scavenger of intracellular NO*,
increasing
oxygen
activities,
rates relative to
cells in the presence of NO*. Furthermore,
had
species
respiratory
ATPase
(51) reported
tempting
protection
with the observed
localized in the close
study
as a
It is therefore
the
of the
uptake
oxygen
cytochrome-c oxidase,
Kaur et al
VHb/VHb-Red.
VHb with the
In this
used to
HMP from NO*. Also
to act
and related molecules would result in the
oxygen
increased
VHb-Red under microaerobic conditions
good agreement
relative to
rate,
higher
NO* radicals than
good agreement,
FHPg-expressing
in
rather
hemoglobin expressing
producing
routinely
in various bacterial
Stevanin et al.
proposed
reduce inhibition of
and
and
respiration by
energy-producing machinery (7).
of
have been
improved efficiency
turnover
(37). Recently,
E. coli cultures
skeletal and cardiac muscles is
to avoid
VHb-R
VHb has been assumed to increase the intracellular
higher
globin-free
proteins
protein production
chain and resulted in
protection
and
of VHb
growth performance
the
to achieve
seem
E. coli cells.
expressing
VHb and
in NO* detoxification
under nitrosative stress conditions do not
growth experiments
significant
hemoglobins
and
flavohemoglobins originating
successfully expressed
115
in the
from various
heterologous wildtype
E.
Chapter 4:
Evidence for role of bacterial
coli MG1655 host. This strain harbors
gene, which
probably provides
due to the induction of the
a
a
hemoglobins
of different
endogenous hmp
chromosomal copy of the
background
endogenous hmp
HMP level in
our
stress
experiments
copy after the addition of the stressors
paraquat and SNP (47, 42). Nevertheless
overexpression
in NO* detoxification
our
and
flavohemoglobins
results
show
hemoglobins
clearly
that
the
increase the natural
resistance of E. coli cells under nitrosative stress.
Acknowledgements
This work
was
supported by
Kelvin H. Lee for the critical
ETH. We would like to thank Susanna Herold and
reviewing
of the
manuscript.
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Gonzalez-Prevatt, V., and D. A. Webster, 1980 Purification and properties of NADHcytochrome o reductase from Vitreoscilla J Biol Chem 255:1478-1482
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Zhao,
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119
Enzyme-independent
Chapter
Expression
(Populus
5:
Transgenic hybrid aspen expressing
of Vitreoscilla
tremula
growth
of
an
x
Vitreoscilla
hemoglobin
tremuloides):
an
in
hemoglobin
hybrid
aspen
attempt to improve
economically relevant tree species
120
Chapter
5:
Transgenic hybrid
aspen
expressing Vitreoscilla hemoglobin
Abstract
We describe in this work for the first time Vitreoscilla
in
an
economically important
tremuloides).
VHb has been
prokaryotic
and
woody plant hybrid
boreal
mainly expressed
with vhb gene
(13)
chlorophyll
enhanced
production.
Similar
of VHb in
a
expression
were
characterized
content,
effects
positive
tobacco
and
a
on
shift
cell
fast
from
(11).
were
generate several transgenic VHb-lines but
between copy numbers and VHb
expression
under different ambient conditions, such
elevated UV-B illumination,
growth improvements
improved growth
did
relative
characteristics
to
be deduced.
the cell structure and cell numbers
were
control
a
lines.
furthermore
latter
finding
source
when
VHb-lines
a
displayed
potential
growth
elevated UV-B illumination
on
are
not
heterologous
stable oxygen
present study,
clear
a
dependence
trend
cell
optimal
number
were
for
and
indicating
was
microscopy, differences
detected between the
VHb lines
expression
Generally, VHb-expression
and electron
higher
nicotine
statistically significant
However,
interest since starch would
conditions
towards
growth temperature
per
increased amount of starch in the
significantly
is of
lines.
high yield,
VHb-expression, analyzed
result in
not
control
can
found.
different
as
generally
plant genotype. Using light
on
was
rates,
In the
no
ATP
papers tobacco
a more
proposed.
of both
higher
observed upon
of VHb is unknown but VHb may contribute to
able to
dependent
anabasine
x
intracellular
to
The mechanism of
level in the cells but other functions have also been
we
leading
germination
were
tremula
organisms
published plant
growth
culture
suspension
by
(Populus
increase
to
of terminal oxidases
activity
levels and ATP turnover rates in bacteria. In the
plants
aspen
in unicellular
suggested
VHb is
eukaryotic origin.
oxygen levels and enhance the
hemoglobin (VHb) expression
provide
transgenic
leaf
chloroplasts.
the
plant
photosynthesis.
interesting
area
a
in
and
and
The
carbon
Effects of
because the lines
responded individually.
Several VHb-lines had elevated levels of total flavonoids,
quercetin-, kaempferol-
and
transgenic
myricetin-derivatives
lines.
121
relative to controls and other
Chapter
Transgenic hybrid
5:
aspen
Vitreoscilla
expressing
hemoglobin
Introduction
Hybrid
aspen is
interspecific
an
and American aspen
characteristics of this
the
hybrid species by
paper
In recent years,
have also
proved
genetic transformation indicating
The
aerobe Vitreoscilla
obligate
limitation.
This
protein
eukaryotic organisms,
cell
growth
known for
and
than
more
where oxygen
a
decade
a
soluble
to
higher
in the ratio of
has been
reported
hypothesized
means
and
for the detoxification of reactive
presumably
also reactive oxygen
al.
germination
rates,
a
(13)
increase
secondary
synthesis
in the
expression of
suggested
cells
on
are
to increase
oxidized
a more
(29). Very recently,
e.g NO and related
VHb in tobacco
plant material,
more
shifts
cytoplasm
it has been
a
compounds,
Similar
VHb in
plants
enhanced
and observed faster
chlorophyll
was
positive
reported
effects
suspension
122
content,
towards
from anabasine
production
02-demanding. Furthermore,
scopolamine production
(7).
expression
hemoglobin proteins might provide
nitrogen,
metabolite
of which is
Datura innoxia with VHb
observed upon
on
VHb
a
species (12,16, 24).
expressed
higher yield
shift in the
nicotine, the
have also
and
prokaryotic
(9,15, 30). Furthermore,
and
E. coli cells
VHb-expressing
under oxygen
of terminal oxidases and thus,
activity
reported
that VHb and other bacterial
Holmberg et
and
for
are
many other
as
higher eukaryotic
In Escherichia coli VHb is
have been
in
heterologous
in bacteria and also in
(15).
well
as
which
on
has been considered to be
ATP levels and ATP turnover rates
NAD+/NADH
aspen
hemoglobin (VHb)
availability
intracellular oxygen levels and enhance the
leading
quality properties,
functionally expressed
been
product yields
superior growth
possibilities for molecular breeding.
the
factor. The beneficial effects of
growth limiting
hybridization.
interest has been focused
special
Hybrid
L.)
competent both for micropropagation and
to be
produces
has
artificial
due to the
due to the wood
industry
tremula
(Populus
aspen
Michx.) produced by
favourable in the manufacture of fine paper.
Populus species
European
have been made
crosses
hybrid.
of
hybrid
tremuloides
(P.
Originally, interspecific
Fi
a
six-fold
upon transformation of
on
growth
were
recently
cultured tobacco cells, where
a
Chapter
shortened
5:
Transgenic hybrid
relative to transformed control cells
but it has been
the
plant
and
proposed
as
reported (11).
positive
our
results
study
VHb-expression
industrial
in
us
changing
to test the
growing hybrid
significance. Hybrid
the
aspen
season.
Thus,
(P.
aspen
as
a
expression. Simultaneously
changes
action in
in
hybrid
scavenging
oxygen radicals,
VHb-technology
during
focused
we
secondary
we
followed
tremula
long-living
acids
fatty
in trees. The
as
is also
tree
life-span
efforts to
study
possible
analyze
exposed
to
well
as
as
the effect of
UV-B illumination
growth characteristics,
metabolites to find out
to
tremuloides) having
x
the whole
our
different cultivation and stress conditions such
and
stable oxygen levels in
generate transgenic VHb-positive lines and
to
different environmental conditions both
during
a more
metabolism
plant
proposed (7).
fast
a
as
cells
It is not known how VHb is
the oxidation of unsaturated
inhibiting
reported prompted
was
VHb-expressing
for
other cellular functions of the
that VHb may contribute to
terminal oxidase,
in seeds have also been
aim of
affecting
were
dry weight
cells. However, other functions, such
functioning
The
expressing Vitreoscilla hemoglobin
and increased final cell
lag-phase
promoting growth
aspen
anatomic
on
VHb-
features,
mechanisms of VHb-
aspen.
Materials and methods
Plant material
Apical
trees
and
axillary
(Populus
Finland
tremula
(61°48'N,
Multiplication
buds derived from suckers of the selected
x
tremuloides), V613, V617,
28°22'E and 61°48'N,
of bud material
modified MS medium
intensity (80-115 pmol
(22)
nv2
was
29°17'E)
were
performed according
under 16:8 h
s-1),
and V619
at 22°C.
123
used
to
hybrid
growing
as
aspen
plus
in southern
expiant
material.
Ryynänen (26) by using
light/dark photoperiod
with the
light
Chapter
5:
Transgenic hybrid
expressing
aspen
Vitreoscilla
hemoglobin
Gene constructs
Disarmed
Agrobacterium tumefaciens
transformation of
and
Bailey (17).
hybrid
vhb gene driven
aspen. The vhb gene has
plasmid pBVHb,
The
by
marker. The construction of
and the correctness of
tobacco transformation
BA and 4
were
5.6,
room
modified MS medium
100
were
were
production
regeneration
verified
as
described
co-workers
includes the
as
selectable
(4).
The
(11)
plasmid
been used in
(13).
clones V613, V617, and V619
designed
for
callus-production
two
an
absorbance of 1 ± 0.2 at 550
plantlets
were
with 0.5 pM
(LB)
After cocultivation the
mg/1
was
was
on
mg/1 kanamycin.
an
cefotaxime before
verified
started
on
was
nm.
(21), pH
with
diluted
Precultured shoot
gentle shaking
expiants,
being
and 300
for 30
by polymerase
From each
Rooting
124
of shoots
cefotaxime.
chain reaction
regenerating
supplemented
if overgrown
transferred to the
mg/1
modified MS medium
MS medium
individual line.
medium
filter paper, and returned onto the callus
on
including 50 mg/1 kanamycin
multiplicated
IAA i.e. to start
days.
rinsed with 500
Agrobacterium
of
in Luria Broth
soaked in diluted bacterial solution with
media
TDZ and 50
chosen to be
pM
was
pM acetosyringone. Overnight culture
medium for
bacteria,
pM
Khosla
neomycin phosphotransferase
VHb-expression vector pBH4, which has
temperature, blotted dry
Elimination of
0.1
vhb gene
overnight with shaking (200 rpm)
pieces
production
callus
pBI121 (Clontech),
by
has been described in Farrés and Kallio
pieces representing
modified MS medium 1:50 to
with
been isolated
genetic
pM 2,4-D for three days before co-cultivation. Single bacterial colonies
containing
min at
used for
transgenic plantlets
on
grown
and leaf
pBVHb
by Holmberg and
Wounded shoot and leaf
precultured
derivative of
was
nopaline synthetase (nos) promoter
amplified
construct is identical with the
Production of
a
originally
35S CaMV promoter and the
gene under control of
(npt2)
(pBVHb)
strain LBA 4404
supplemented
callus
with 2.22
was
(PCR)
one
pM
shoot
and
with
was
BA and 2.85
performed
on
growth
Chapter
5:
Transgenic hybrid
regulator-free MS medium.
in
a
aspen
The rooted
expressing
hemoglobin
Vitreoscilla
plantlets were transferred to
soil and grown
greenhouse.
Analysis
PCR
of the transformed lines
used for
was
presence of
samples
(10),
and vhb genes. For PCR
nptl
were
taken and
with minor modifications
used
amplify
to
bp
150 mg callus
analyses
was
according
619
a
DNA
genomic
isolated
to Aronen
as
described
or
in vitro shoot
Doyle
Häggman (2).
and
of
fragment
aspen lines for the
hybrid
the different
preliminary testing
the
nptl
Doyle
and
The
primers
5'-
were
gene
TGGGCACAACAGACAATCGG-3' and 5'-CAGCAATATCACGGGTAGCC-3', and
the
for
specific
primers
437
a
of
fragment
bp
the
vhb
5'-
were
5'-
and
ATGTTAGACCAGCAAACCATTACC-3'
TCAACCGCTTGAGCGTACAAATCT-3'. Genomic DNA isolated both from the
plantlets
used
as
templates (0.5-1.0 pg)
Furhermore,
confirmed
absence
the
using
in the PCR
as
described
pair
primer
of the bacterial
(bases 1315-2932)
Genomic DNA for Southern
(5'-CACTCTTCGGAAGTAGTGGG-3',
5'-
dissolved in TE buffer
(10
samples
RNase. Genomic DNA
transformed with
separated
transferred to
pBVHb
on
a
a
0.8%
nylon
Germany) by capillary
(437
bp)
were
mM
were
in
for
a
716
bp fragment
was
of the
occ
region
isolated from the leaves of the
the method of Lodhi et al.
et al.
(30). Finally,
the
(19)
further modified
precipitated
DNA
was
Tris-HCl, 1 mM EDTA, pH 8.0), and treated with
from the nontransformed control lines and lines
digested
(w/v)
with Hindlll.
agarose
membrane
gel by
(Boehringer
transfer. Double stranded
labeled
regenerated
the
Ti-plasmid (32).
by Valjakka
and described in detail
Häggman (2).
was
hybridization
greenhouse-grown plants using
Aronen and
by
were
material
Agrobacterium
of
AAGCTATGCCGAACATCGGG-3'), specific
were
(i.e. negative controls)
of transformed and nontransformed lines
with
Twenty-pg
standard
probes
of
samples
procedures (27)
Mannheim GmbH,
digoxigenin-11-dUTP.
125
DNA
and
Mannheim,
nptl (619 bp)
and vhb
Prehybridisation
and
Chapter
hybridisation
Transgenic hybrid
5:
performed
were
°C with both vhb and
0.1% SDS at
detected
Briefly,
8.0),
protein
membrane
supplied by
respectively.
rabbit
(11),
The VHb
Buckinghamshire, UK)
chlorophyll
For
or
et
al.
pi
of the
(5).
(25).
measured
by
albumine
as
serum
gels
The
detected
ECL-based
used
by
at
pH
proteins
and
Sambrook
antirabbit
(Amersham
blotting
(27),
al.
et
generated
in
immunoglobulins
Biotech,
Pharmacia
(Finnzymes, Espoo, Finland)
western
according to the manufacturer's instructions.
and
protein
(50
were
homogenized
mM MES, 20 mM
mg frozen
100
measurements
in
liquid nitrogen
MgCb,
50 mM
used for
The rest of the extract
was
centrifuged (20'800
pi
supernatant
The amounts of
was
used for soluble
chlorophylls
and
protein
protein
126
assay
were
sample
assay
min)
described
measured
the
1% TWEEN 80,
according
g, 4°C, 5
as
from
and diluted in 5 ml
/3-MeOH,
chlorophyll
was
200
standard. The
VHb antiserum
of the extract
x
(13).
the method of Bradford
electrophoresis
and
(18)
by HRP-conjugated goat
were
described
and blotted onto Immun-Blot PVDF
Laemmli
were
Either
as
NaCl, 0.2 mM EDTA (pH
in 0.1 M
(Hercules, CA).
by
analysis
protein assays
of extraction buffer
2
was
ChemiGlown-based
uppermost full-size leaves
pH 6.8).
supernatants
monomers
detection systems
and
were
twice at 14000 rpm for 15 minutes. The
centrifuged
described
as
and visualised
Chlorophyll
washed
PMSF, 2% PVP and 50 mM Tris buffer
1 mM
Bio-Rad
(Bio-Rad, Hercules, CA).
blotting
were
twice at 68°C for 15 min with 0.5x SSC
homogenized
SDS-PAGE
15%
on
performed
were
the membranes
hybridisation,
NaCl, 0.3 M sodium citrate, pH 7.0) containing
reagent and bovine
Bio-Rad
separated
were
were
concentration of the
(5) using
was
ß-mercaptoethanol,
homogenates
8.0. The
at 42
(Boehringer Mannheim)
solution
studied with Western blot
was
200 mg leaf material
1 mM
hemoglobin
to the manufacturer's instructions.
of vhb gene
Expression
M
Vitreoscilla
digoxigenin-labeled hybridisation products
The
SDS.
according
After
temperature, and
room
0.1%
containing
(3
expressing
Easy Hyb
in
nptl probes.
twice for 5 min with 2x SSC
aspen
using
to Porra
and 2
by
x
200
Bradford
UV-2401 PC
Chapter
Shimadzu
5:
Transgenic hybrid
spectrophotometer
calculated
according
expressing Vitreoscilla hemoglobin
aspen
and the amounts of
chlorophylls
a
and b
were
(1).
to Arnon
Microscopy and morphometric analyses
For
light and
electron
the middle of
6%
fully expanded
glutaraldehyde
in the
7.2, rinsed
Dialux 22
same
microscope.
of 10 000 with
Relative cell
area
parenchyma layers
transgenic hybrid aspen
Philips
buffer
lines
phosphate
were
number
were
and
mesophyll
a
transgenic lines.
grid
was
with lines
Epon (modified
placed
cells
with lines
used for
spaced
cells
representing
Plus 1.4
used
were
to
1.0 pm
with
a
cell
area
to
in
the
cytoplasm
and
at
the
(33).
using
Randomly
proportions
of
of control lines
chloroplast
pm at the
to 30'000X
0.133/pm
palisade
sections
analyze
in
optical
both control lines and
program.
corresponding to 0,667
corresponding
Leitz
microscope.
analysis
cytoplasm
micrographs magnified
at 4 mm,
(23)).
random block orientations,
cut at
measured from transverse
Image-Pro
at 20 mm,
electron
over
from
photographed
per relative
For determination of total
spaced
from
in 1% Os04 in 0.1 M sodium
mitochondria, chloroplasts, starch and plastoglobuli
and
cut
MgCb, pH
containing
208 transmission electron
mesophyll
microscope
of
a
chloroplast
of leaf
mm were
and lead citrate, and recorded at electron
acetate
and
1
mm x
1 mM
Ultrathin sections
magnification
areas
of 0.5
stained with toluidine blue, and
uranyl
chosen
phosphate
in ethanol and embedded in
were
BK51
samples
leaves outside the mid vein, and fixed for 4 hours in
buffer, postfixed
stained with
Olympus
studies
in 0.05 M sodium
buffer, dehydrated
thick sections
microscopic
volumes
specimen level,
A square
specimen level,
grid
was
morphometric analyses
of
plastoglobuli
and mitochondria. Four to 12
from each of three
or
four
of
micrographs
control lines
were
controls and 1400
analyzed.
/ pm2
The total
for the
samples
cytoplasm
transgenic
lines.
127
area
transgenic lines
analyzed
was
as
well
as
2020/pm2 for
Chapter
5:
Transgenic hybrid
aspen
expressing
Vitreoscilla
hemoglobin
Secondary metabolites
Phenolics
ice
were
on
0.6 ml of 100% cold methanol and
by adding
homogeniser
with maximum
ice for 15
homogenate
transferred
The
centrifuged
after
step with
s.
were
combined, methanol
samples
were
stored at -20°C until
was
a)
into
was
evaporated
allowed to stand
6 ml
repeated
under
more
nitrogen
and
(%)a)
Solvent B
Too
ö
5
100
0
10
80
20
20
70
30
30
65
35
40
50
50
45
50
50
Rinsing
0
100
Equilibration
100
0
1.5%
tetrahydrofuran+
0.25%
and the
times. The
finally
the
gradient elution for phenolics.
ö
Aqueous
was
Supernatant
glass-vial
3
s
analyzed.
Solvent A
(min)
was
with Ultra-Turrax for 30
centrifugation
Table 1. The conditions used in HPLC
Time
homogenate
on
with Ultra-Turrax
for 3 min at 13'000 rpm.
0.6 ml methanol
extracts
Eppendorf tubes
homogenizing
homogenization
was
immediately
extraction
previous
for 30
speed
min, after which
and the
repeated
was
extracted from 5-10 mg of dried leaf material in
(%)
acid in DAD and acetic acid in MS
orthophosphoric
b> 100% methanol
For HPLC
analyses
analyzed by
the
samples
DAD-HPLC.
(positive ions) (14)
was
were
Retention
used to
HPLC is described in Table 1. The
on
commercial standards
(+)-catechin, chlorogenic
quercetins
on
on
or
and
UV-spectrum
time,
identify
of the
(1:1)
and then
HPLC/API-ES
and
the components. The
LC-quantification
gradient
components
used in
was
based
purified components. Salicin, salicortin, tremulacin,
acid based
hyperin, kaempferols
chlorogenic acid,
dissolved in methanoLHzO
on
on
neochlorogenic
standards; Myricetins based
kaempferol-3-rhamnoside,
acid
128
on
chlorogenic
acid.
on
myricitrin,
cinnamic acids
Chapter
Greenhouse
5:
Transgenic hybrid
aspen
experiments
established
were
of the vhb gene and its effect
expression
Vitreoscilla
hemoglobin
experiments
Greenhouse
morphology
or
of
production
transferred into
a
mixture of
and forest soil
an
and
integration
growth characteristics, anatomy,
on
different cultivation
experiments the
pots and acclimatized
plantlets
in vitro
at
were
(Kekkilä, Finland),
unfertililized and Ca-free peat
in 0.141
(2:2:1)
stable
study
to
secondary compounds during
conditions. Before the initiation of the
perlite
expressing
high humidity for
two
weeks.
The first
greenhouse experiment
greenhouse experiment
The first
designed
was
transgenes by Southern hybridizations and
blot
analyses
growth
in
transgenic aspen
characteristics
humidity
the
plants
commercial 0.2 %
started
decreased to take
planted
Superex
after
place
experiment photoperiod
For
humidity
the
included.
Thus,
to
was
represented
all three
greenhouse.
was
20°C at
day
extra
hybrid
transgenic
lines with five
experimental design
consisted
aspen clones and 15
randomly assigned
aspens of the
dry weight and
high
in
one
month
fertilization
experiment.
was
was
In this
and 18°C at
night.
than 40%.
same
age to
or
of
blocks. The
274
V619)
plants
plantlets,
and 50 control
V619).
replicates
were
equalize light conditions
129
more
were
which
VHb-expressing lines (7 lines
lines from clone V613,19 from clone V617 and 14 from clone
two
for
Thereafter
until the end of the
clone V613, 7 from clone V617 and 1 from clone
divided into
Western
maintained for 16 h with SON-T AGRO 400 W Na-
the
hybrid
of
pots and daily fertilization with
transfer
day
integration
period
acclimatization
(Kekkilä, Finland) lasting
was more
the
fresh and
fertilizer
every third
experiment only
morphology,
into 0.5 1
lamps (Philips AB). Average temperature
Relative
confirm stable
study VHb-expression by
After
examined.
were
were
month
one
lines. Also
to
to
The
from
plants (17
plants
were
surrounded
for all
plants.
by
Chapter
The
in both
and
started
experiment
beginning
Transgenic hybrid
5:
on
replica were randomly
per VHb
or
For leaf
analyses
were
and leaf
were
taken
measured
6 leaves per
using
video
a
and
dry weight
plants
were
and
during
growth
the
and 5
plants
a
designed
was
growing
season
characteristics
fertilized with 0.2%
plants
connected to
camera
greenhouse experiment
metabolism
assays
were
per
9% N, 3,5% P, 5% K, 3.8% S, 4.8%
0.04% Zn, and 0.015% Mo.
Kemira
Mg,
humidity
Photoperiod
For this
experiment
was more
we
lines
growth
represented by
analyses,
collected
on
transgenic
to follow
for
fresh
or
November 17.
control line
and to
VHb-expression
study anatomy.
examined. In this
period
Agro Oy, Finland)
and
Also fresh
the
experiment
(Kekkilä, Finland)
and then
5
was
planted
in
kg/m3 including
maintained for 16 h with HPI-T 400 W
22°C at
was
day
and 18°C at
selected four VHb-lines 3, 5, 18 and 28
characteristics
representing
replicate)
night.
than 40%.
V613 and four lines 45, 51, 60 and 62
the best
on
0.04% B, 0.08% Mn, 0.05% Cu, 0,05% Fe,
lamps (Philips AB). Average temperature
Relative
per line per
commercial fertilizer
5-Superex
peat (Kasper,
collected
were
graphic TV monitor.
twice at the end of the one-month-acclimatization
commercial fertilized
samples
of leaves, stems and roots, the number of
plant
The second
at least three
samples,
protein
control lines
6 and 20, November 2
November 2. Southern blot
on
greenhouse experiment
November 17. At the
VHb-expressing and
size leaf from 4
The second
secondary
on
analyzed from the samples
area were
analysis
area
finished
regular (October
and
chlorophyll
weight and dry weight measurements
twigs
from all
selected for
samples (one full
November 2. Leaf
side
was
and diameter measurements. Leaf
control line, for
Western blot
expressing Vitreoscilla hemoglobin
October 6 and it
experiment five plants
of the
17) height
aspen
representing
during
the first
both V613 and V617 clones
48 individual
plants
and
they
assigned blocks.
130
representing
clone
clone V617. These lines showed
greenhouse experiment.
were
were
Control
also included. Each line
divided into four
was
randomly
Chapter
The second
5:
Transgenic hybrid
greenhouse experiment
13th of October. At the
and controls in four
expression studies,
replicate)
were
October. For
studies leaf
of the four
replica
were
leaf
on
on
11th of
were
replicates representing
VHb-lines
experiment
aim
secondary
of the
in the
experiment
was
on
metabolism. At the
greenhouse experiment.
V613/5
beginning
(Kasper,
kj
at first acclimatized for
subsequent
the
of the
plants
nr2 per
one
three weeks
day
kj
week
on
not
V617/60
as
well
from
as
part of the
leaves in the upper
which
London, UK)
lamps
were
stress
were
nr2 per
on
day.
emitted
remove
some
were
as
131
week
nr2 per
plants
day
kj
on
and
nr2 per
with the natural, local UV-B
was
provided by Phillips
UV-C radiation emitted
a
one
The other half of the
kj
in
in the second
elevated UV-B illumination level 7.2
UV-A radiation
planted
three weeks
subsequent
UV-B illumination 3.6
corresponds
UV-B
when the buds had not
at first acclimatized for
and then for
by
matter content and
started, the plants
covered with cellulose-diacete film
in order to
effect caused
experiment,
illumination level at the end of June. The radiation
UV-B
analysis samples
selected from each
Agro Oy, Finland)
Kemira
The ambient UV-B level selected
lamps,
and
VHb-expression, growth, dry
ambient UV-B illumination level 3.6
day.
and 10th of
September
plants randomly
study
to
Half of the
UV-B illumination 1.8
then for
25th of
per line per
plants
greenhouse
commercial fertilized peat
were
14 and 28,
11 and October 10. For anatomical
fully developed
yet and the elongation growth had
flushed
from all VHb-lines
for Western blot
as
collected from four
illumination and its effect
on
well
as
finished
plant were sampled.
stem per
The
September,
was
measurements. For VHb-
full size leaf from 4
days August 14, September
samples
and it
regular (August
selected for
randomly
control lines V613 and V617. Two
UV-B
August
experiment five plantlets
the
samples (one
collected thrice
hemoglobin
Vitreoscilla
started 1st of
was
secondary compound
collected
were
beginning of
expressing
25, October 10) height and diameter
11 and
September
aspen
polyester
(0.115
by
mm, FilmSales
the
film
UV-B
lamps.
(0.125
Ldt,
Because the
mm,
FilmSales
Chapter
Ltd)
A
over
The
used to
was
the
5:
Transgenic hybrid
give
from the second
i.e. VHb-lines
representing
randomly assigned
lasted for
one
elongation
blocks. This
month
ending
up
dry weight analyses
weights, chlorophyll
relative cell
and
area
and
test.
they
amount of UV-
were
were
were
used for this
Leaf
samples
measured
on
Proportions
April
were
growth
and it
collected and
23 and
and leaf, stem and root
May
7. Leaf
experiment May
7.
dry
secondary compounds
as
made
by
per relative cell
compared by
for
samples
measurements, fresh and
area were
the Student-Neuman-Keuls
mitochondria,
of
April
derived from the top
analyses
amounts, amount of
means were
divided into two
established 10* of
for Western blot
chloroplast number
of variance, and the
range
was
May.
data from
protein
and
collected at the end of the
were
comparisons among
multiple
7* of
secondary compound analyses
Statistical
analysis
on
plants
analyses
as
plants
experiment
plant
Statistical
well
same
greenhouse experiment
10 individual
and diameter of all
for
fresh and
the
3, 5, 18 and 28 representing clone V613, lines 45, 51, 60
and from the middle of the
samples
plants
hemoglobin
clone V617 and control lines from V613 and V617 clones. Each
represented by
was
Vitreoscilla
experiments.
experiment,
line
expressing
to both the control and UV-B
one-year-old plants
and 62
aspen
chloroplasts,
starch
and
plastoglobuli were analyzed using Student's t-test.
Results
Production of
Hybrid
case
of
lines
transgenic
aspen clones differed in their competency for
clones
independent,
V613, V617 and V619
PCR
positive,
lines two turned out to be
nptl.
The escape lines
and
transformation. In the
produced altogether 14,
regeneration capable lines, respectively.
partly
were
we
genetic
escapes,
including only
omitted for Southern blot
132
24
and 4
From these
the selectable marker gene
analyses
and
subsequent
Chapter
5:
Transgenic hybrid
greenhouse experiments
only.
Stable
lines
representing
integration
aspen
and the lines with
of both
nptl
4
9
16
c
68
c
hemoglobin
good regeneration ability
clones V617, V613 and V619,
18 28
5
Vitreoscilla
and vhb genes
number of transgenes varied from 1 to 4
3
expressing
was
were
selected
confirmed in 12, 8, and 1
respectively (Figure 1).
The copy
(Figure 1).
45 51
60 62
49 63 64
c
kb
8.6
Ü
-.>
3.6
1.9
Figure
1. Southern
with
vhb
a
probe.
analysis
pBVHb
transformed
Lines numbered 3-16
V619 and lines 45-64
representing
of
represent clone
(Populus
V617. C
=
analyses
were
greenhouse experiment
the Western blot
expressing
A.
non-transformed control
tremuloides) lines
68
represents clone
plants
from left to
right
performed
greenhouse experiment
four weeks after the initiation of the first
at the time when all lines
analyses
all tested
VHb and the
were
still
4
«•É
5
growing. According
plants (three randomly
expression level
was
line
chosen
9
m
16
28
-
gag
45
plants / line)
V619
V617
+
49
51
60
62
68
* ëM' «•*«
133
to
dependent (Figure 2A).
V613
3
x
tremula
clones V613, V617, and V619.
Western blot
+
aspen
represent hybrid aspen clone V613, line
Characterization of VHb-lines in the first
were
hybrid
-
Chapter
B.
5:
Transgenic hybrid
aspen
expressing
Vitreoscilla
V617
V613
3
1
3
1
2
3
+
-
1
-
controls
51
45
controls
5
2
hemoglobin
2
12
3
3
+
-
-
<*»••
3
Aj
E,
A2
Ai
E2
A2
Ei
E2
+
-
controls
18
controls
5
A}
-
A2
Ei
E2
+
-
-
fgß
Figure
2. Western blot
VHb
A.
protein.
analyses
expression
indicate that all tested
was
experiment
at the time when the
dependent.
B.
growing
VHb-expression
during
season
26.9. and 3
=
plants
for
subsequent three
were
plants
then for
plants
was
still
were
on
ambient
mature leaf close to the
on
(A)
greenhouse
elongation growth
lines
the
end
of
the
5>18>28>9>3>4>16>control.
2
=
on
on
thinner
in
Figure
(p< 0.05)
were
one
week
on
turned out to be line
plants throughout
VHb expression
kj
were
experiment
in
The
3. At clonal level the
plants
and
nr2 per
day.
kj
kj
than the other clones. There
diameter within the clones.
134
was no
=
of the tested
the
clone
were
1
plant.
ranking
was
significant
of V617
and then
day
V613
in
day
nr2 per
followed the
ranking
=
half
UV-B illumination 3.6
clone
12.9., 2
kj m-2per day. The other
mature leaf located in the middle of the
of the lines
=
the
studied. Half of
was
nr2 per
UV-B illumination level 7.2
(E)
greenhouse
taken thrice 2
UV-B illumination 1.8
60>45>control>2>51>62>64>49>control>5>63. The
presented
grown
UV-B illumination level 3.6
elevated
top of the plant,
When the
week
one
at first acclimatized for
three weeks
able to express VHb
are
growing. VHb-expression
followed in the
at first acclimatized for
subsequent
at
were
greenhouse experiment. Samples
the second
weeks
VHb-lines
studied 4 weeks after the initiation of the first
10.10. C. Also the effect of UV-B illumination
the
of the
transgenic
following:
V617
was
differences found
shorter
(p< 0.05)
are
and
statistical difference in stem
Chapter
At the end of the
leaf, shoot and
statistically
expressing
5:
Transgenic hybrid
experiment, fresh
root
lower
fresh
(p< 0.05)
lines had
soluble
differences
proteins,
was
leaf
area
the
were
found in
or
clone V617 and V619
only
plants.
DVHb/3
DVHb/28
DVHb/18
4>
20
O
o
VHb/5
DD Control/3
J3
10
0
weeks
50
B)
JVHb/63
aVHb/49
?40
u
BVHb/64
DVHb/62
DVHb/51
£
30
M
<u
20
o
o
5
aVHb/45
VHb/60
a
-
in the
weight
case
values relative to control
of clone V613
content
ID Control/5
Control/2
10
weeks
135
or
(p< 0.05).
No
in the amount of
in the number of side branches. In leaf
no
significant
differences
were
found
significantly bigger (p<
level, leaf
in clone V617 than in clones V613 and V619.
IVHb/9
d
VHb-
At clonal
IVHb/4
30
Generally,
VHb-line 3 had
IVHb/16
40
measurements indicated that
were
chlorophyll
50
A)
hemoglobin
corresponding dry weights
and fresh
significant only
than in the control
(p< 0.05)
smaller
Vitreoscilla
in clone V617 than in the other clones.
among lines and within clone V613
0.05)
and
in the number of leaves
measurements in
area
weights
expressing
dry weight
and
higher dry weight
lines but the difference
significant
aspen
size was,
significantly
Chapter
5:
Transgenic hybrid
aspen
expressing
Vitreoscilla
hemoglobin
C)
weeks
Figure
3. Increase in shoot
were
followed and
were
performed
length
compared
of
transgenic
to controls
during
VHb-lines of
the first
at two weeks intervals October 6 and
hybrid
aspen
(P.
tremula
greenhouse experiment.
x
The measurements
20, November 2 and 17. A. Lines of clone
V613. B. Lines from clone V617 and C. lines from clone V619. Different letters indicate
differences within the
Second
sampling
tremuloides)
significant
time.
greenhouse experiment
to follow
VHb-expression
and to
study anatomy
and secondary metabolism
VHb-expression
was
measured 4, 6 and 8 weeks
experiment. Expression levels
Variation in the
after
did not vary between the
expression level was found
within lines.
4
weeks
136
the
initiation
of the
sampling days (Figure 2B).
Chapter
140
5:
Transgenic hybrid
expressing
aspen
Vitreoscilla
hemoglobin
n
DVHb/51
DVHb/62
120-
VHb/45
1T
be
VHb/60
1°°-
u
DD Control/3
5
80-
leng
o
Sho t
ODD Contro/4
o
a
20-
a
be'
be
i—(HiBhïïiilïïii
n
0-
i
mi
8
6
4
2
0
weeks
Figure
were
length
4. Increase in shoot
followed and
experiment
compared
of
to controls
the cultivation conditions
measurements
were
performed
VHb-lines of
transgenic
during
were
the second
hybrid
at two weeks intervals
(P.
tremula
greenhouse experiment
than
optimal
more
aspen
August
during
the
14 and 28,
tremuloides)
x
in 2000. In this
previous
September
October 10. A. Lines of clone V613. B. Lines from clone V617. Different letters indicate
differences within the
When the
sampling
ranking
in clone
was
V617
are
conditions
optimal
experiment
were
and
no
of the lines
was
followed the
ranking
5>28>18>control>3. The
following
the
25,
significant
of the tested
corresponding
significant
60>45>control 4>control 3>62>51. The
was
differences found
11 and
time.
elongation growth
lines in clone V613
year. The
presented
the
in
Figure 4.
plants
significant
In this
were
experiment
than in the
taller
differences
clonal
when the cultivation
in
first
greenhouse
elongation growth
were
observed.
The concentrations of
kaempferol-dervivatives
significantly decreasing
(Figure 5).
in both clones
sampling
variations in the contents of
during
the
dates
secondary
as
apigenins
(Figure 5).
was
of
the
and thus
no
metabolites.
137
phenolic
was
experimental period
The two clones showed
general
acids
significantly decreasing
salicylates, quercetin-derivatives
experimental period
these types of
well
(613, V617) during
Furthermore the content of
between the first two
as
only
minor
and total flavonoids
trend could be observed for
Chapter
5:
Transgenic hybrid
Vitreoscilla
expressing
aspen
hemoglobin
Apigenins
Salicylates
b
*-
b
J3
-,
T a
-
M
4
a
X-xx
-
%
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3
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b
•^.
M
E
1
-
r\
c
15.8.
12.9.
c
12.9.
15.8.
10.10.
10.10.
Total flavonoids
Quercitin-derivatives
£0
-
-**
J3
20
er
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-
abB---x<
'S
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15
-
10
-
u
o
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^
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c
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d
5
M
E
n
15.8.
12.9.
.
15.8.
10.10.
10.10.
12.9.
Phenolic acids
Kaempferol-derivatives
JA
JSf
'8
^tJ?
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b4
o
Sl
7
3
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-
b
-
C
2
1
n
15.8.
12.9.
15.8.
5. Intraclonal fluctuation in
secondary
period
of
x
indicate
aspen
significant
significant
are
VHB-lines 8.8 ±1.0
(P.
tremula
(p< 0.05)
tremuloides) clones
found in
and XX
figure by
(V613
X
(
V413
(p< 0.05)
quercetin-derivatives
also indicated in the
mg/g dw)
12.9.
10.10.
metabolite concentrations
intra- and interclonal differences
differences
and control lines
-
10.10.
Figure
hybrid
-
(V617
138
and V617
at different
and in
(d).
mg/g
experimental
Different letters
sampling
apigenins
times. The
between VHb-lines
control line 13.5 ±1.9
control line 4.1 ±0.4
mg/g dw), respectively.
)
the
during
mg/g
dw
>
V617
dw> V613 VHb-lines 3.4 ±0.2
Chapter
5:
Transgenic hybrid
Controls and VHb-lines differed
The concentration of
cases:
±1.9
(13.5
sampling
(4.1
±0.4
mg/g dw)
aspen
in the
significantly (p< 0.05) only
quercetin-derivatives
than in V617 VHb-lines
time and the concentration of
mg/g dw)
hemoglobin
Vitreoscilla
expressing
(8.8
apigenins
(3.4
than in V613 VHb-lines
±
mg/g dw) during
±1.0
was
0.2
two
in V617 control lines
higher
was
following
the first
in V613 control line
higher
mg/g dw) during
the second
sampling time.
Analysis
of one-pm-sections, stained with toluidine blue, revealed
differences in leaf anatomy between the cross-sections of the
leaves and control leaves. Beneath the upper
larger
than in the lower
The cells below the
epidermis,
epidermis
there
were
epidermal cells,
and formed
the lower cells. However, in VHb-lines cell size in
smaller than in controls
transgenic
layer
Chloroplast
than in the outer
number
one
was
(p< 0.05)
higher
but
palisade parenchyma
was
in the inner
significant
a
loose
spongy
parenchyma
of
was
similar in both
was
higher
cell
palisade parenchyma
differences between VHb-lines
and control lines could not be found. About half of the leaf
occupied by
were
compact layer than
lines and controls this indicates that the total number of cells
in VHb lines.
VHb-line
where the cells
a more
Because the leaf size
(p< 0.05).
transgenic
no
two-cell-layer-palisade parenchyma.
was
larger
generally
cross
cells
small
and
section
was
conspicuous
intercellular spaces.
Table 2.
Morphometric
determinations of volumes of mitochondria,
starch and
Material
na>
plastoglobuli
for control and VHb-lines of
Percentage
of
cytoplasm
Mitochondria13)
Peroxisomes
Chloroplastsb)
peroxisomes, chloroplasts,
hybrid
aspen.
Percentage of chloroplasts
Starchc>
Control-lines
68
2.61 ± 0.39
0.64 ± 0.21
68.5 ± 2.47
11.6
VHb-lines
51
3.18 ± 0.59
0.42 ± 0.19
68.6 ± 1.97
20.4 ±4.28*
+
3.18
Plastoglobuli0)
0.94 ±0.14
1.10 ±0.12
micrographs taken from two different control lines and VHb
P < 0.05.
lines, each line containing 3 or 4 different plants.
b) Mitochondria and
chloroplasts are expressed as means ± SE of total cytoplasmic volume.
c) Starch and
plastoglobuli are presented as means ± SE of the corresponding chloroplast volume.
a) n
=
number of measured electron
*
=
139
Chapter
In
depth
studies
interesting
Transgenic hybrid
5:
ultrastructural
on
occupied
the
was
almost
(Table 2). However,
grains
the
and covered
a
larger
but these differences
larger
was
peroxisomes
not
in
not
VHb-lines
was
higher
cytoplasm.
observed
Figure
in
RER
the
in
was
of
m,
as
micrographs
of
and in the control leaf cells
and
was
individual
compared
were
than in control
also
to
bigger
chloroplasts,
corresponding
the
as
well
single
as
lipid droplets
between
cells of
grains
hybrid
in
relatively large (B).
mitochondria; p, plastoglobulus;
s,
were
starch;
v,
a
different
aspen
(P.
vacuole;
w, cell
were
visible
were
lines.
tremula
chloroplast (A)
Mitochondria
of
value
cisternae. No differences
control line has several small starch
140
chloroplasts
percentage volume of mitochondria
mesophyll
Lim.
single,
determinations
in VHb-lines when
organelles
are
a
VHb-lines, but these differences
bodies
these
transgenic VHb-line chloroplast starch grains
chloroplast;
morphometric
chloroplasts. Furthermore,
controls
present only
cells had
some
cytoplasm. Chloroplasts
thin
transgenic
The
in controls than in
6. Transmission electron
cristae. c,
Mesophyll
chloroplasts
significant.
than
occurrence
tremuloides). Non-transgenic
and
relatively large
volume in VHb-line
were
in
a
cells revealed
of starch in the VHb-line
significant. Only occasionally Golgi
in the
mesophyll
(Figure 6). Osmiophilic plastoglobuli
in control lines
ones
large
than in control
to be
by
cytoplasm
as
volume
appeared
also
of the
exactly
significantly (P<0.05) bigger
those
of
morphology
surrounded
was
largest portion
showed that it
starch
expressing Vitreoscilla hemoglobin
differences between VHb and control lines.
central vacuole that
large
aspen
are
x
while in
rich in
wall. Bar
=
0.5
Chapter
5:
Transgenic hybrid
aspen
expressing
Vitreoscilla
hemoglobin
Effect of UV-B illumination
lines
Transgenic
regardless
of the
in the middle of the
line
transgenic
of the
analyses
Table 3.
transgenic
in
ambient
and
plant.
(Fig. 2C).
Variation in the
For instance,
weak whereas the
Significant
VHb and of
same
UV-B
consistent
expiant leaf,
of the
position
elevated
The
VHb-expression (Fig. 2C).
consistent
found
both
i.
the
levels
was
in line
VHb-expression
in
V617/45
differences found in the contents of
non-transgenic hybrid
expression
e. near
expression
expression
illumination
was
showed
level
was
top of the plant
dependent
V613/3
was
on
or
the
in most
mostly good.
secondary compounds
in leaves of
aspen cultivated under ambient and elevated UV-B
illumination3)
Growth conditions
Secondary compound
groups
Ambient UV-B
Elevated UV-B
Apigenins
none
none
none
Catechins
none
Condensed tannins
none
Kaempferol-derivates
none
Myricetin-derivates
none
Phenolic acids
Quercetin-derivates
V617/62: 8,5
V617/45: 4,1
±
1,3
±
0,8
none
V617/60:1,4 ± 0,lc>
V617/45:1,7 0,1*»
V617/control: 0,5 ± 0,1 V617/control: 0,5 ± 0,1
V617/62: 0,7 ± 0,1
V617/62: 0,7 ± 0,1
±
0,7 ± 0,1
0,7
0,1
V617/control:
V617/control:
±
±
0,6
0,1
0,8
0,2
V617/60:
V617/45:
± 0.0
0.0
±
0,1
V617/control:
V617/control: 0,1
V617/control: 0,2 ± 0,1 V617/control: 0,2 ± 0,1
V617/ 62: 0,2 ± 0,0
V617/ 62: 0,2 ± 0,0
V617/51: 0,3 ±0,1
V617/51: 0,3 ± 0,1
±
none
V617/51: 3,5 ± 0,5
V613/5: 9,5 ± 1,6
V613/3: 3,0 ± 0,4
Salicylates
V613/18:111,7 ± 17,4
Total flavonoids
V613/5: 231,7 15,2
V613/28: 248,0± 24,0
V613/3: 3,9 ± 0,5
V617/60: 8,8 ± 0,7
V617/45:10,9 ± 1,5
±
control:
3,2
0,3
V617/control: 3,2 ± 0,3
V617/
±
V617/control: 5,7 0,9 V617/62: 4,1 ± 0,3
V617/51:5,4±l,2
V617/62: 4,1 ± 0,3
none
±
V613/5:ll,
± 1,8
V617/60:10,9 ± 0,9
V617/control: 3,8 ± 0,3 V617/control: 3,8 ± 0,3
V617/62: 5,1 ± 0,4
V617/62: 5,1 ± 0,4
V617/45:13,4
7 ±1,9
V617/51:6,7±l,4
V617/control:6,7±l.l
a)
Significant differences in experimental groups display P < 0.05.
Groups of lines, within which statistically significant differences have been determined.
c> Concentration of
secondary metabolites is given in mg/g dry weight, means and standard
are given.
b)
141
error
Chapter 5: Transgenic hybrid
When
considering
growth
the
V617/45 (22.8
than
(9.9
control line
2.9
±
all lines in both treatments
leaf and shoot
treatment
and this
analysed.
were
was
significantly (p
also the
In fresh and
VHb-line had
flavonoids than VHb-lines
values
significant
V617/45
<
0.05)
only significant
case
better
when
dry weight percentages
of
statistical differences either within the
no
was
grew
in elevated UV-B
between the treatments.
especially V613/5
the
cm)
± 3.4
only
illumination, however, VHb-lines responded individually.
In UV-B
B
or
cm)
there
samples
expressing Vitreoscilla hemoglobin
data within the treatment
illumination the line
one
aspen
did
not
were
V617/60
and
salicylates, quercetin-derivatives
V613/18, V613/3
differ
differences
more
had
from
the
generally
and
controls.
only
found
In ambient UV-
and total
V613/3, respectively (Table 3)
In
elevated UV-B
illumination
in lines of the clone V617. VHb lines
more
quercetin-derivatives, kaempferol-
derivatives, myricetin-derivatives and total flavonoids than the controls
V617 VHb lines
but
or
other
(Table 3).
Discussion
In this paper
we
for the first time
are
in
tremula
tremuloides).
an
organisms
of both
generated
vhb
increased
growth
nicotine with
positive
were
prokaryotic
a
to
no
aspen
mainly expressed
plants showing
tobacco
dry
matter. In
reduced
(Populus
in unicellular
et al.
(13)
germination time,
addition, higher production of
concomitant decrease in anabasine content is detected in VHb-
plant
of
relative to non-transformed control
suspension
cultures derived from
transformed control cultures has been
able to generate several
numbers but
hemoglobin (VHb)
eukaryotic origin (15). Holmberg
and
rates and content of
improved growth
We
This far VHb has been
carrying
tobacco
plants relative
Vitreoscilla
economically important boreal woody plant hybrid
expression
x
describing
clear
dependence
transgenic
plants. Furthermore,
VHb-expressing
reported
lines with
very
recently (11).
slightly varying
between copy numbers and
142
tobacco
copy
VHb-expression
Chapter
5:
Transgenic hybrid
levels could be found.
VHb-expression
was
the copy number has direct effect
found to be
was
Expression
of VHb did not vary
hemoglobin
in lines with 3
stronger
in
experiments.
between
elongation growth and
differences
significant
Some
transgenic
lines
observed in
VHb-expressing
either
on
hybrid
of the lines.
or
leaf
expressed.
were
between
recorded
finding
were
is in clear
(13) showing growth promoting
tobacco. Our results revealed that
possibility
some
transgenic
lines
characteristics relative to control lines. However,
that the effect of transgene
integrational position
if
VHb-
general growth improvements
et al.
Holmberg
predict
could not find any clear correlation
elongation growth
no
to
sampling day
lines relative to control lines. This
highly improved growth
cannot exclude the
we
to the
4 gene
or
Generally,
genetic background
the
the amount of VHb
in
contradiction to the results of
effects in
addition,
(Figure 3). However,
VHb-expressing
on
possible
level.
VHb-expression
significantly according
In
position
our
the
on
dependent
expression
of the vhb gene
or on
the
expression
is
we
dependent
genotype of the
various
aspen lines.
The effect of different cultivation conditions, i.
on
Vitreoscilla
Due to the limited amount of this kind of lines it is not
copies.
show
expressing
aspen
growth
characteristics of
within
single
size, i.
e.
clonal
backgroud,
a
relative to the 2nd
are
line
experiment,
Na-lamps
average temperature
was
the end of the second
experiment.
growth
of
well
One
as
more
which
lines was, however, obvious. Differences in
prominent
was
in the first
are
we
elaborate
lamps
growth
the cultivation
instead of
regular
greenhouse experiment. Furthermore,
during the light period.
was
The shoot
length
at
almost doubled relative to the first
that under the
not
more
optimized
and HPI-T multimetal
season
might speculate
greenhouse experiment
conducted under
used in the first
growing
greenhouse experiment,
between the different lines from the identical
increased 2°C
VHb-expression
1st and 2nd
greenhouse experiment
using pre-fertilized peat
fertilization and
effects
as
markedly
conditions. For the second
conditions
transgenic
e.
optimized
culture conditions the
observable, whereas under less permissive
conditions differences would become
143
more
obvious. Our
unpublished
Chapter
results
clearly
5:
Transgenic hybrid
show that the effect of
homozygous VHb-positive
conditions such
under
as
unpublished).
Clearly,
and
these
an
In
expressing
Vitreoscilla
VHb-expression
plants
are
on
in contradiction to the work of
addition,
protein
content
are
differences
no
were
our
in contradiction to the
been
(7),
Plants
species.
protective pigments,
such
produce
might
with two-years old
apigenins.
plants
between control lines and VHb-lines could found.
differences in
When
a
changes
few
or
at the
secondary
V617 VHb-lines. However, this
metabolite levels
effect
on
the
and thus
no
been
In the UV-B
differences in
of other
morphological (number
only significant
were
analyzed VHb-expressing
exposed
to UV-B illumination the
In elevated UV-B illumination
had
more
was
not
a
especially
quercetin-derivatives, kaempferol-
general
would like to propose that VHb-lines may
against
set
cases.
V617/45 and V617/60
themselves
as
of
reactive radicals
derivatives, myricetin-derivatives and total flavonoids than the controls
we
as
shown).
it has
Analyses
characteristics revealed
area)
generally responded individually.
VHb lines
no
growth (fresh weight, dry weight)
lines did not differ from the control lines. When
lines
Since
significant
shoot
of leaves and side branches, leaf
not
generation
to UV-B radiation.
experiment
as,
the
highly
illumination
parameters such
such
VHb work in tobacco
trough
buffer these
exposed
were
no
A.D. and Kallio P.T.,
experiments (data
previous
(13),
upon the exposure to UV-B radiation
that the presence of VHb
VHb-lines and control lines
length
of
reported (13).
flavonoids and
as
et al.
characteristics,
other
in
obvious in
chlorophyll content has
increase in
hypothesized
growth properties
Holmberg
(Frey,
UV-B illumination is known to induce oxidative stress
of
the
obvious under various stress
only
differences could be observed
findings
reactive oxygen
hemoglobin
reduced temperature, oxidative and nitrosative stress, whereas
statistically significant
where
tobacco
optimized conditions,
chlorophyll
aspen
oxidative stress. It
plant cell,
changes
i.
in
.e.
VHb
might
secondary
seems
bind
use
different mechanisms to protect
possible
/ detoxify
production
144
other
increase in all lines and therefore
that VHb has
a
protective
the reactive oxygen
metabolite level would be induced
illumination. Thus the level of flavonoids
or
is not increased
by
species
the UV-B
significantly.
Chapter
5:
Transgenic hybrid
This
suggestion
and
flavohemoglobins
various
is also
part of the
are
microorganisms (12, 20, 34).
flavonoids
transgenic
(such
lines
We may
against
of
the
control
materials
either
by
an
utilization.
the
to
of
of starch
photosynthesis),
synthesis
carboxylase
function
Interestingly,
aimed at to
the
of
more
the
or
in
the
By targeting
in the
between
could be
triggered by
accumulation
of
higher
energy
starch
and
carbon
ratio is
and trees. This mechanism links
supply
of
photosynthate (i.e.
Thus,
it
to the
might
of
cycle
that the
possible
carboxylase
/
than in the control lines
(6).
al.
(8)
photosynthesizing activity by targeting
the
(3)
chloroplasts.
and
Chaparro-Giraldo
It has been
the oxygen avid Lba to the
chloroplast
improve
145
et
hypothesized
that the
by changing the CO2/O2
which thus, could prevent the wasteful
oxyxgenase reaction and therefore
be
(ribulose-l,5-bisphosphate
of Barata et al.
efficiency
in
explained
Pi/3-phosphoglycerate
with the
oxygenase reaction of Rubisco could be controlled
chloroplasts,
number
and therefore results in
prominent in the VHb-lines
soybean leghemoglobin (Lba)
the
higher
exchange
improved
an
plants
is abundant.
Rubisco
experiments
improve
these
because the enzyme is activated when the Calvin
3-phosphoglycerate
enzyme is
of
of those VHb-
ability
exchange
enhanced
prevailing opinion
intermediate
oxygenase)
better
starch content of VHb-lines could be
ADP-glucose pyrophosphorylase activity
products
This
chloroplasts
in
measure
Higher
to the
principal regulator
increased level of
protection
metabolic
indicate
enhanced carbon fixation
According
oxidative stress in
experimental cytoplasmic area)
chloroplasts.
and
able
of VHb-lines.
chloroplasts
hemoglobins
lines relative to VHb lines.
photorespiration in control
were
an
increased
an
indicating
from the total
higher phosphoglycolate production
we
against
response
ultrastructural measurements, that the
on
peroxisomes, mitochondria,
Interestingly,
that bacterial
protection.
peroxisomes (calculated
cytoplasm
findings
hemoglobin
On the other hand
UV-B illumination,
based
speculate,
stress
kaempherols) suggests
as
lines to allocate energy to
of
the recent
supported by
Vitreoscilla
expressing
aspen
ratio in
photorespiration activity.
the authors assumed to
impair
carbon fixation rate. The results in
Chapter
tobacco revealed
levels
involved
generates
(8).
Transgenic hybrid
5:
differences in
no
deleterious effects
even
Both groups
expressing
on
hemoglobin
(3). Surprisingly,
plant growth
that the
suggested
Vitreoscilla
photosynthesis, starch,
metabolism
aerobic
in
aspen
the
and enzyme
Lba-expression
production
in
potato
effects could be due to either
observed
insufficient Lba concentrations in the
and tuber
sucrose
chloroplasts
or
inappropriate O2 binding
kinetics of Lba.
An
efficiency
metabolic
improved
and energy household in VHb-lines could also
result in the accumulation of starch.
increased
levels
ATP
(29). Interestingly,
limitation
as
(28).
this effect
increased substrate-level
potentially
suggested
by promoting glycolytic
An increased
growth
beginning
or
conditions, where the de
accumulated
starch
cellular activities and
expression
of
the
that
flux
wildtype
non-
charge,
maize cells under oxygen
non-symbiotic hemoglobins accomplish
through
resulting
NADH oxidation,
in
phosphorylation.
starch content
favor the
either at the
constitutive
the total ATP level, relative to
The authors
has been shown to result in
in maize cells resulted in enhanced energy
symbiotic barley hemoglobin
determined
VHb-expression
of
as
observed
hybrid
aspen at
at end of the
novo
would
the
in
lines would
non-optimal growth conditions,
growing
synthesis
VHb-expressing
season or
of carbon
under
sources
e.
g
non-optimal light
is restricted and the
help transgenic VHb-expressing plants
to
sustain
growth.
References
1.
Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta
vulgaris.
Physiol. 24:1-15.
and
H. Häggman. 1995. Differences in Agrobacterium infections in silver birch
Aronen, T.,
and scots pine. Eur. J. Forest Pathol. 25:197-213.
Barata, R. M., A. Chaparro, and S. M. Chabegas. 2000. Targeting of soybean leghemoglobin
in tobacco chloroplasts: effects on aerobic metabolism in transgenic plants. Plant Science
Arnon, D. I. 1949.
Plant
2.
3.
155:193-202.
4.
Bollinger,
C.
microaerobic
J., J.
E.
growth
Bailey,
and P. T. Kallio.
2001. Novel
hemoglobins to enhance
Prog. 17:798-
and substrate utilization in Escherichia coli. Biotechnol.
808.
146
Chapter
5.
protein Utilizing principle
8.
9.
aspen
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Saccharomyces
J Biol Chem 271:25131-25138
148
cerevisiae
-
Evidence for
a
role
Conclusions
Conclusions and final remarks
In the last few year research
gained
tasks. Indeed
they
other functions than pure oxygen
serve
able to
are
perform catalytic
is
dependent
integrated
into the
same
were
in the
Vitreoscilla
proteins,
promote growth of
for several years to
known to lack oxygen
However,
case
detoxification
also of VHb, it
activity
might
observed effects
VHb
into
on
account
reported
the
of bacterial
various
growth
and
about VHb and
oxygen to the
proposed
VHb
inhibitory
it
might
action of NO.
regarding growth promotion
finding might be
is
an
was
dependent on a catalytic reaction,
be
a means
as
149
organisms
and
explain
the
that VHb
Instead of
to
delivering
detoxify
NO and
would be
cytochromes
a
might
reductase domain to
flavohemoglobin (VHb-Red)
enhanced relative to VHb. This
growth promoting
such
NO
the various effects of
plausible
By engineering
significantly
indication that the
reported
them with the latest results
of the
the C-terminus of VHb, the effect of this artificial
yielding
pathways.
from various
flavohemoglobin.
activity
which
to increase
energy
which
findings,
might provide
the
more
the anaerobic
connecting
has been
heterologous hosts,
productivity. Considering
shown for E. coli
respiratory chain,
from the
flavohemoglobins
the model of VHb action to
adapt
nitrogen species. Therefore,
protected
recent
most
flavohemoglobins,
as
over
flavohemoglobin
be necessary to
protect the cytochromes,
reactive
be favored
the metabolism of E. coli and
on
of
might
proposed
This
hemoglobin VHb,
VHb has been
during growth.
pathways would
taking
flavohemoglobins
storage and delivering
effective intracellular oxygen concentration and therefore the
aerobic metabolic
in
protein.
One member out of this group of
expressed
and
has
widespread
nitrogen species.
system, which
reductase
on a
flavohemoglobin
activities and their main task
be the detoxification of NO and related reactive
function is
and
hemoglobins
bacterial
prokaryotic organisms. Surprisingly,
have been shown to
hemoglobin
has been shown to be
protein family
This
momentum.
bacterial
on
effect of VHb
/
VHb-Red
the NO detoxification reaction.
Conclusions
VHb and VHb-Red have been demonstrated to be able to
clearly
shown for VHb-Red and to
indicate
the
cytochromes
VHb and
preferred
it
localization of VHb in the
close
in both Vitreoscilla and E. coli cells. However,
cytochromes
Altogether
extent also for VHb.
some
seems
that VHb has
and in the
reactive
nitrogen species
noxious
compounds. Therefore,
out that VHb is able to
Moreover,
has been
recent data
neighborhood
a
of the
direct interaction of
has not yet been shown.
likely
effect of VHb is based
cells from the
degrading activity
effects of nitrosative stress. Furthermore, NO
negative
protect
on
its
serve
it
an
important
protection
can
protective
be
role in the detoxification of
of the cellular metabolism from these
hypothesized
that the
growth supporting
role. However it cannot be
other functions, such
150
as
oxygen
completely
delivery.
ruled
Curriculum vitae
Curriculum vitae
Alexander Daniel
Citizen of
Frey,
March 3rd, 1974
Auenstein, AG
Education
April,
1981
April,
1986
August,
-
-
March 1986
Primary school, Kiitttigen,
June, 1990
Bezirksschule Aarau
1990
-
June, 1994
Kantonsschule Aarau,
AG
Typus
B
Studies
October, 1994
March, 1998
-
-
March, 1999
October, 1998
Study of Biology
Technology.
Diploma
Thesis at the Institute of
the group of Prof.
June, 1999
-
April,
2002
at the Swiss Federal Institute of
Biotechnology
in
J. E. Bailey.
Research assistant and Ph D Thesis at the Institute
of
Biotechnology,
Technology in the
Bailey.
Swiss
Federal
research group
Institute
of Prof.
J.
of
E.
hemoglobin (VHb) to a
novel class of protective and growth promoting
bacterial hemoglobin proteins"
Thesis: „From Vitreoscilla
List of
publications
Frey,
D.,
and
A.
Bailey, J. E.,
engineered
Escherichia coli.
and P. T. Kallio. 2000.
Vitreoscilla
Appl.
Expression
hemoglobin-reductase
fusion
Envrion. Microbiol. 66:98-104.
151
of
Alcaligenes eutrophus flavohemoprotein
protein
for
improved hypoxic growth
of
Curriculum vitae
Frey,
A. D.,
Fiaux, J., Szyperski, T., Wüthrich, K., Bailey, J. E., and P. T. Kallio. 2001. Dissection of
central carbon
resonance
metabolism
flux distribution
of
hemoglobin-expressing
analysis
in microaerobic
Escherichia coli
bioprocesses. Appl.
by
13C
nuclear
magnetic
Envrion. Microbiol. 67:680-
687.
Frey,
A.
D., Rimann, M., Bailey, J. E., Kallio, P. T., Thompson, C. J., and M. Fussenegger. 2001. Novel
pristinamycin-responsive expression systems
for
plant cells.
Biotech.
Bioeng.
74:154-163.
Kallio, P. T., Frey, A. D., and J. E. Bailey. 2001. From Vitreoscilla hemoglobin (VHb)
growth stimulating hemoglobin proteins.
eukaryotic
Frey,
cells. A
A. D.,
comparative view
Farrés, J.
Bollinger
C.
on
In: Recombinant
host
J. T., and
physiology.
to
a
novel class of
protein production with prokaryotic
and
Kleuwer Academics Publishers, pp. 75-87.
P. T. Kallio. Bacterial
hemoglobins
and
flavohemoglobins
for alleviation of nitrosative stress in Escherichia coli. In press.
Häggman, H., Aronen, T., Frey,
A. D.,
Ryynänen, L., Julkunen-Tiitto, R., Timonen, H.,
Pihakaski-
Maunsbach, K., Chen, X., and P. T. Kallio. Expression of Vitreoscilla hemoglobin in hybrid aspen
(Populus
species.
tremula
x
tremuloides):
Submitted for
an
attempt
to
improve growth
publication.
152
of
an
economically
relevant tree