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Khadijah
Suhaimi
Peterhouse
Overall the aim was to explore the mechanisms of mul.drug transporter lmrP and to see if it t proposed model of transport* (gure 6) LmrP is a plasma encoded H+ an3port transport protein in Lactococcus Lac.s. We inves3gated: 1. If Calcein, Calcein.AM or EtBr are substrates for export and if there is substan3al eux 2. The requirement of metabolic energy for transport through produc3on of pmf (p) 3. The importance of Asp-235 and Glu-327 acidic residues in facilita3ng transport 4. If Calcein is ac3vely taken up by LmrP
CLEAVAGE
Types of Cells: Hydrophilic + Non Polar (Acetomethoxy deriva3ve) i. WT LmrP ii. DE mutant LmrP - muta3on in Asp-235 (D) and Glu-327 (E) iii. 8048 (Control no LmrP) Figure 1 1. Eux of Calcein.AM and Calcein Fluorometer was used to measure uorescence of calcein (non-membrane permeable) (gure 1) in cells and supernatant. Centrifuge to separate. Readings were taken at 0, 10 and 20 mins a^er Calcein.AM or buer (control) added. Amount of calcein recorded over 3me. 2. Eux of EtBr and Growth Rate of Lactococcus Lac.s Spectrometer was used to measure op3cal density of the dierent cells. All cells made up to 0.1 absorbance. Measurements taken every hour to observe growth rate. 3. EtBr Eux Assay of Ini.ally De-energised Cells EtBr intercalates in DNA and uoresces. Fluorometer used to measure amount of EtBr in cell. Glucose added a^er set 3me for EtBr satura3on (vary for each cell type).
25
Is
Calcein.AM
a
substrate
for
export
by
LmrP?
DE
m utant
Calcein.AM
Figure
2
shows
WT
and
DE
Mutant
Calcein-AM
15
LmrP
have
the
same
uorescence
level
DE
mutant
but
less
than
control
indica3ng
some
DMSO
10
8048
Calcein- eux.
Absence
of
LmrP
results
in
AM
Calcein
accumula3on.
Calcein.AM
5
8048
DMSO
eux
further
supported
in
Figure
3
-
DMSO
solvent
as
nega.ve
control.
0
uorescence
was
no3ceably
larger
in
non-expressing
LmrP
cells
sugges3ng
Time
(mins)
eux
of
Calcein.AM
out
of
cell
by
Figure
3:
Time
Dependence
of
control
LmrP
before
it
has
3me
to
be
cleaved
400
Calcein
Eux
from
Cells
(8048)
cells
(Ac.on
shown
in
gure
1).
350
Is
there
a
substan.al
eux
of
Calcein
control
300
(8048)
by
LmrP?
supernatant
250
We
found
there
was
no
signicant
LmrP
cells
dierence
of
Calcein
(error
bars)
in
200
Cal-AM
cells
compared
to
supernatant.
150
Fluorescence
levels
(Calcein)
remained
LmrP
100
supernatant
steady
in
LmrP
expressing
cells.
Cal-AM
50
Calcein
levels
decreased
in
control
cells
which
may
be
due
to
endogenous
0
0
min
10
min
20
min
Calcein
transporter
in
L.
Lac3s.
Time
(min)
20
Fluorescence (A.U)
Fluorescence (a.u)
1 13 25 37 49 61 73 85 97 109 121 133 145 157 169 181 193 205 217
EtBr
which
is
a
intercala3ng
mutagen
inhibits
growth
of
L.Lac3s
through
inducing
muta3ons.
If
cells
are
able
to
extrude
EtBr
this
will
increase
growth
rate.
High
op.cal
density
=
greater
number
of
cells
=
Higher
growth
rate.
Therefore
WT
LmrP
must
be
ac3vely
extruding
EtBr.
4:
Lactococcus
Lac.s
Growth
Figure
D.E
Mutant
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Time
(h)
0
In
Presence
of
EtBr
Control
Wt
LmrP
DE
Mutant
LmrP
shows lihle dierence to control (no LmrP). This indicates that Asp-235 and Glu-327 might be necessary for EtBr eux.
We
now
know
from
gure
4
that
EtBr
is
substrate
of
LmrP.
Is
metabolic
energy
required
for
transport?
EtBr
enters
cells
through
facilitated
diusion,
intercalates
into
DNA
and
uoresces.
Upon
glucose
addi3on
in
WT
LmrP
shows
immediate
eux
highligh3ng
need
for
ATP
for
produc3on
of
pmf
(p).
DE
Mutant
shows
lower
rate
of
eux.
This
indicates
DE
residues
are
sucient
for
eux
but
not
essen3al.
Control
lacks
EtBr
Glucose
Figure
5:EtBr
Eux
Assay
with
250
LmrP
but
ATP
LmrP
powers
H+
Glucose
200
Glucose
ATPase
which
pumps
H+
out
150
crea3ng
nega.ve
100
wtLmrP
poten.al
which
Demutant
LmrP
could
ahracts
50
8048
EtBr
in
cell
0
leading
to
an
increase
of
EtBr
SATURATION
entry.
Time
(s)
Fluorescence
(A.U)
1
87
173
259
345
431
517
603
689
775
861
947
1033
1119
1205
1291
1377
1463
1549
1635
Our data (gure 4+5) suggests that LmrP is able to extrude EtBr (+vely charged). This concept ts Figure 6 whereby low anity, nega3vely charged, hydrophobic binding pocket of LmrP is facing inwards. Opposite charges allows interac3on and subsequent binding. Figure 5 also suggests metabolic energy is required in the form of pmf as well as a H+ gradient. Proposed model suggests high anity outward facing side necessita3ng the need for pmf (H+ an3port) to power substrate release. H+ associa3on could indicate pH dependent transport as it will inuence protonated state of residues and thus binding poten3al to substrate. Further experiments would need to be conducted to be certain. However mutant in gure 5 s3ll shows eux. DE residues are not necessary for transport. This is further reinforced in gure 2 with Calcein.AM eux in DE mutant LmrP being similar to WT LmrP. However as EtBr was not extruded to the same extent it could be said that EtBr is more dependent on DE residues. Calcein.AM and EtBr are clearly a substrates for eux but Calcein was not shown to be. The ques3on remains if LmrP is capable of substrate Figure 6: Proposed mechanism inux. Interes3ngly EtBr satura3on is fastest in WTLmrP. (gure 5) for transport by LmrP.