1
Supp
orting
Information for
Bioenergetic suppression by redox
-
active metabolites promotes
antibiotic tolerance in
Pseudomonas aeruginosa
Richard D. Horak
1
, John A. Ciemniecki
1
, Dianne K. Newman
1,2
,*
1)
Division of Biology and Biological Engineering, California Institute of Technology,
Pasadena, CA 91125
2)
Division of Geological and Planetary Sciences, California Institute of Technology,
Pasadena, CA 91125
*
Dianne K. Newman
Email:
dkn@caltech.edu
This PDF file
includes:
Figures
S1
to
S
8
2
Figure S1.
Chemical structures of endogenous RAMs tested.
3
Fig
ure
S
2
.
1 hour RAM treatments in PBS do not
dramatically alter the dimensions of cells or
conflate the DiOC
2
(3) results of Figure 1D. (A) ECDFs of side
-
scatter
-
channel values of events
used to plot Figure 1A. Cell suspensions treated with RAMs for 1 hour in PBS before DiOC
2
(3)
staining do not dramatically differ in size. (B) ECDFs of DiOC
2
(3) Red/Green fluorescence values
used to plot Figure 1D normalized by their side
-
scatter
-
channel values. Each condition contains
~50,000 events. Differences in cell dimensions does not account for the changes in ΔΨ
observed
following RAM treatments.
4
Figure S
3
.
Treatment of cell suspensions with PYO arrests cell doubling. Representative phase
-
contrast images of cells within a Cell ASICS chamber with or without PYO
treatment at 0 minutes
and 100 minutes post PYO treatment. Scale bar = 5 μm.
5
Figure S
4
.
PYO protects WT cell suspensions like
Δphz
suspensions, moving cells into anoxia
lower tobramycin killing, and RAMs, CCCP, or nigericin do not affect cell survival in the absence
of antibiotics. (A) PYO and PBS incubation affects the tobramycin tolerance of WT PBS cell
suspensions like
Δphz
suspensions.
“No Wait” suspensions were pre
-
treated in PBS with 100 μM
PYO for 1 hour then given 100 μg/mL tobramycin for 2 hours. “Wait 1 Hour” suspensions were
incubated in PBS for 1 hour, then treated with 100 μM PYO for 1 hour, then given 100 μg/mL
tobramyci
n for 2 hours.
(B) Cell suspensions moved into anoxic conditions survive
2 hour
tobramycin
treatments
more than those left in oxic conditions during treatment. (C) PCA, PCN,
PYO, TOX, CCCP, and nigericin do not affect the survival of cells in the absence of antibiotics.
(A, B, C) Log
-
transformed CFU/mL values of biological replicates. Error bars represent
95%
confidence intervals. (A) Shows 4 biological replicates per condition. (B, C) Shows 3 biological
replicates per condition.
6
Figure S
5
.
Solvents used in RAM stocks did not dramatically alter DiOC
2
(3) Red/Green
fluorescence ratios or survival to tobramycin. (A) ECDFs of DiOC
2
(3) red/green fluorescence
ratios following 1 hour treatment with PBS (No RAM), DMSO, HCl,
NaOH, or CCCP. Equimolar
amounts of solvents used in RAM treatments did not dramatically change the observed ΔΨ
values. Each conditions contains ~50,000 events. (B) Log transformed CFU/mL values of cell
suspensions pre
-
treated in PBS for 1 hour with, PYO,
HCl, DMSO, NaOH, or no treatment, before
addition of tobramycin for 2 hours. While PYO
–
solubilized in 20 mM HCl
–
increases survival to
tobramycin, equimolar amounts of solvents used in RAM treatments do not protect cells against
tobramycin.
7
Figure S
6
.
RAMs produce unique ΔΨ subpopulations over time. Histograms of 3 biological
replicates of DiOC
2
(3) Red/Green fluorescence ratios of PCA, PCN, PYO, TOX, or CCCP treated
cells at 15
-
, 30
-
, 45
-
, or 60
-
minutes post
treatment. Values normalized to the mean fluorescence
ratio of the 15
-
minute No RAM condition. Each histogram line contains 50,000 events.
8
Figure S
7
.
Succinate synergizes with tobramycin while glyoxylate protects against it, PYO
synergizes with colistin, unlike tobramycin or ciprofloxacin. (A) Treatment of
MOPS media
cell
suspensions with succinate increases tobramycin lethality and abolishes PYO’s protective effect
while glyoxylate increases survival. (B) PYO does not protect and lightly synergizes with colistin.
(A, B) Log
-
transformed CFU/mL values of biological rep
licates. Error bars represent 95%
confidence intervals. (A) 3 biological replicates per
condition. (B) 4 biological replicates per
condition.
Antibiotic treatments were done for 2 hours following a 1
-
hour pre
-
treatment by addition
of 100 μg/mL of tobramycin, or 10 μg/mL of colistin to suspensions.
9
Figure S
8
.
ABBAs have a steep O
2
gradient, with O
2
hitting undetectable levels within 300 μm of
the ABBA surface. Representative biological replicate. Line represents the mean of 3 technical
replicate measurements taken within seconds of one another with an O
2
micro
-
electrode probe.
Circles represent the 3 technical replicates for each depth.