VS
H526Y
x8
Create populations
polymorphic for antibiotic
resistance mutation.
Propagate for 30 days in rich, drug free, m
edia. B
ottleneck every 24h.
Analyse and fit m
athematical
model to dynam
ics of more
costly resistance.
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24h
24h
…
Figure 1
H526D
H526Y
H526D
x8
VS
H526Y
x8S531F
H526Y
S531Fx8
VS
x8S531F
S531Fx8
K43T
K43T
Figure 1. Testing the evolvability of different antibiotic resistances.
y = -0.0744x - 0.1557
-10
-8
-6
-4
-2 0 2 0 50
100 150
200 250
300
Ln(H526Y/H526D)
s H526Y - s H526D
t H526Y - t H526D
A
BC
Figure 2
y = -0.0744x - 0.1557
-10
-8
-6
-4
-2 0 2 0 50
100 150
200 250
300
Ln(H526Y/H526D)
Figure 2. High evolvability of costly H
526Y allows its long-term
maintenance
y = -0.015x - 0.5875 -3
-2
-1 0 1 2 3 4 5 6 0 50
100 150
200 250
300
Ln(H526Y/S531F)
s H526Y - s S531F
t H526Y - t S531F
A
BC
Figure 3
Figure 3. Different evolvabilities betw
een Rifam
picin resistance alleles
y = -0.0319x - 0.6438 -7
-5
-3
-1 1 3 5 7 9
11 0 50
100 150
200 250
300
Ln(K43T/S531F)
s K43T - s S531F
t K43T - t S531F
A
BC
Figure 4
y = -0.0319x - 0.6438 -7
-5
-3
-1 1 3 5 7 9
11 0 50
100 150
200 250
300
Ln(K43T/S531F) Figure 4. Differences in evolvability betw
een Rifam
picin and Streptomycin resistance alleles
-0.09
-0.04
0.01
0.06
0.11
0.16 rpoA (T196I)
rpoC (R1224C)
rpoA (T196I), waaZ (S280P)
yifE (G39D)
rpoC (S486P)
rpoC (H450P)
Selective Effect
vs Ow
n Background (H
526Y)
vs Com
petitor Background (S
531F)
AFigure 5
B
Figure 5. Fitness effects of the evolved mutants in the com
petition between H
526Y and S531F
-0.09
-0.04
0.01
0.06
0.11
0.16
rpoC (K50T)
nrfG/gltP nadK/recN, yedW(IS1) znuA (F37L), sspA (L156P)
paoB (V231E)
yeaR (IS186)
envR (IS5)
Selective Effect
vs Ow
n Background (S
531F) vs C
ompetitor B
ackground (H526Y
)
Mutation in Resistance 2
Background (NR2 )
Fitness WN
R2 = 1.027 Tim
e TN
R2 = 3
Relative Effect = �
s = WN
R2 - W
R2 = 0.027
!R
elative Time = �
t = TN
R2 - M
aximum
Time = - 276
Experimental
Inferred
Time
Time
AB
LNResistance 2
Resistance 1( )LNResistance 2
Resistance 1( )
WR1 =1
WR2 =0.96
Mutation in Resistance 2
Background (NR2 )
Fitness WN
R2 = 1.057 Tim
e TN
R2 = 3
Mutation in Resistance 1
Background (NR1 )
Fitness WN
R1 = 1.068 Tim
e TN
R1 = 29
Relative Effect = �
s = (WN
R2 - W
R2 ) - (W
NR
1 - 1) = 0.029!
Relative Tim
e = �t = T
NR
2 - TN
R1 = -26
Experimental
Inferred
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WR1 =1
WR2 =0.96Figure S1. E
xample of the fitting process for a sim
ulated experimental population.
y = -0.015x - 0.5875 -3
-2
-1
0
1
2
3
4
5
6
0 50 100 150 200 250 300 Ln(H
526Y
/S53
1F)
y = -0.0744x - 0.1557
-10
-8
-6
-4
-2
0
2
0 50 100 150 200 250 300
Ln(H
526Y
/H52
6D)
A
B
C
Figure S2. Long-term dynamics with the identification of the fluorescent backgrounds
y = -0.0319x - 0.6438 -7
-5
-3
-1
1
3
5
7
9
11
0 50 100 150 200 250 300 Ln(K
43T/
S53
1F)
−7
−5
−3
−101
3
5
7
9
11
0 25 50 75 100 125 150 175 200 225 250 275Generations
LN(K43T/S531F)
−3
−2
−1
0
1
2
3
4
5
6
0 25 50 75 100 125 150 175 200 225 250 275Generations
LN(H526Y/S531F)
−10
−8
−6
−4
−2
0
2
0 25 50 75 100 125 150 175 200 225 250 275Generations
LN(H526Y/H526D
)
A
B
C
Figure S3. Long-term dynamics from the inferred parameters for each replicate population
y"="0.0424x")"0.0903"R²"="0.03072"
!0.4%
!0.3%
!0.2%
!0.1% 0%
0.1%
0.2%0%0.1%
0.2%0.3%
0.4%0.5%
0.6%0.7%
0.8%0.9%
1%
/itness"effect"
initial"frequency"
Test"for"negative"frequency"dependent"selection"
Figure S4
Figure S4. Test for negative frequency dependence selection in competitions
between resistance strains H
526Y and H526D
H526D&(R1)H526Y&(R2)
Ini/al&H526Y&Frequency
W&
TW&
T
Popula/on&10.538
1.57611
1.55215
Popula/on&20.382
1.29311
1.26322
Popula/on&30.488
1.38741
1.34851
Popula/on&40.468
1.48326
1.4432
Popula/on&50.413
1.42121
1.40930
Popula/on&60.476
1.47431
1.47842
Popula/on&70.513
1.21811
1.1919
Popula/on&80.493
1.77618
1.76723
Popula/on&90.557
1.0173
Popula/on&100.461
1.02829
Popula/on&110.581
1.0233
Popula/on&120.58
1.00246
1.023
Popula/on&130.523
1.02314
Popula/on&140.55
1.00138
1.02823
Popula/on&150.59
1.66837
1.67145
Table 1. Evolutionary Param
eters estimated for the com
petition between strains H
526Y and H526D
. W
stands for the fitness of the emerging haplotype and T its tim
e of appearance. Initial Freq stands for the inferred initial frequency of the H
526Y background. In the cases where only one of the backgrounds has acquired a
mutation, the inferred param
eters are in bold for the background where no m
utation was inferred.
S531F&(R1)H526Y&(R2)
Ini1al&H526Y&Frequency
W&
TW&
T
Popula1on&10.369
1.45915
1.47217
Popula1on&20.458
1.04146
1.0513
Popula1on&30.277
1.43615
1.4519
Popula1on&40.472
1.0347
1.0544
Popula1on&50.286
1.5298
1.55412
Popula1on&60.454
1.58555
1.63760
Popula1on&70.381
1.61536
1.65540
Popula1on&80.325
1.46511
1.49115
Popula1on&90.473
1.19919
1.19414
Popula1on&100.378
1.16314
1.15410
Popula1on&110.356
1.59517
1.60119
Popula1on&120.483
1.0283
1.0494
Popula1on&130.37
1.34411
1.34912
Popula1on&140.444
1.5429
1.56212
Popula1on&150.446
1.50516
1.51417
Popula1on&160.468
1.64755
1.759
Table 2. Evolutionary Param
eters estimated for the com
petition between strains H
526Y and S531F
. The meaning of the param
eters is as in Table 1.
Table 3. Evolutionary Param
eters estimated for the com
petition between strains K43T and
S531F. The m
eaning of the parameters is as in Table 1.
S531F&(R1)K43T&(R2)
Ini1al&K43T&Frequency
W&
TW&
T
Popula1on&10.505
1.20716
1.1883
Popula1on&20.319
1.34920
1.35926
Popula1on&30.299
1.54618
1.54721
Popula1on&40.349
1.2616
1.25318
Popula1on&50.319
1.6694
1.7375
Popula1on&60.359
1.4574
1.47410
Popula1on&70.333
1.08852
1.0634
Popula1on&80.256
1.3257
1.33412
Popula1on&90.43
1.68737
1.73242
Popula1on&100.335
1.0973
1.094
Popula1on&110.357
1.53750
1.52953
Popula1on&120.388
1.1611
1.1446
Popula1on&130.41
1.1955
1.1937
Popula1on&140.379
1.09543
1.0586
Popula1on&150.329
1.66325
1.67230
Table 4. Potential com
pensatory mutations identified in the genom
es of the clones evolved in the competition betw
een resistances H
526Y and S
531F.
Background
Genom
e Position
Gene(s)
Mutation
Annotation
FrequencyFunction
3,438,465rpoA
C→
TT196I
51.5%R
NA polym
erase, alpha subunit4,184,828
rpoCT→
CS
486P25.5%
4,187,042rpoC
C→
TR
1224C6.1%
4,184,721rpoC
A→
CH
450P2.9%
3,797,382w
aaZT→
CS
280P19.7%
Lipopolysaccharide core biosynthesis protein3,946,224
yifEG→
AG
39D12.0%
Conserved protein, U
PF0438 fam
ily, unknown function
4,292,389nrfG
/gltPA→
TIntergenic
5.0%[nrfG
] Hem
e lyase (NrfE
FG) for insertion of hem
e into c552, subunit N
rfG/[gltP
] glutamate/aspartate: proton sym
porte3,637,091
pitAC→
TA
476V2.6%
Phosphate transporter, low‑affinity; tellurite im
porter
374,196m
hpE/m
hpTT→
CIntergenic
2.4%4‑hyroxy‑2‑oxovalerate/4‑hydroxy‑
2‑oxopentanoic acid aldolase, class
I/putative 3‑hydroxyphenylpropionic transporter
3,464,375bfr
C→
TR
125C2.0%
Bacterioferritin, iron storage and detoxification protein
1,743,180ydhQ
T→C
S
324S3.1%
Hypothetical protein
3,898,043yieL
C→
AT186T
2.5%P
utative xylanase4,183,521
rpoCA→
CK
50T14.8%
RN
A polymerase, beta prim
e subunit
4,292,389nrfG
/gltPA→
TIntergenic
19.6%[nrfG
] Hem
e lyase (NrfE
FG) for insertion of hem
e into c552, subunit NrfG
/ [gltP
] Glutam
ate/aspartate:proton symporte
2,749,808nadK
/recNΔ
1 bpIntergenic
18.1%[nadK
] NA
D kinase/ [recN
] Recom
bination and repair protein
1,940,499znuA
T→C
F37L17.7%
Zinc transporter subunit: periplasmic‑binding com
ponent of AB
C
superfamily
3,374,976sspA
T→C
L156P12.2%
Stringent starvation protein A
300,420paoB
T→
AV
231E11.8%
PaoA
BC
aldehyde oxidoreductase, FAD‑containing subunit
2,036,728yedW
IS1 Ins
Coding
9.9%P
utative DN
A‑binding response regulator in two‑com
ponent system w
ith YedV
1,877,853yeaR
IS186 Ins
Coding
8.4%H
ypothetical protein3,410,893
envRIS
5 InsC
oding6.8%
DN
A‑binding transcriptional regulator2,361,326
yfaY A→
GV
110V9.4%
Hypothetical protein
S531F
RN
A polymerase, beta prim
e subunit
H526Y
!
Table S1. Fitness costs im
posed by the antibiotic resistance alleles (K43T, S531F, H
526Y, H526D
) measured in com
petition against the sensitive reference strain.!!!!!
Antibiotic!Resistance!
Target!Genes!Am
inoacid!change!(location)!Fitness!cost!
(relative!to!wild:type)!
Standard!Err!K43T%
Streptomycin!
rpsL%AAA!:!ACA!(128)!
0.092%0.034!
S531F%Rifam
picin!rpoB%
TCC!:!TTC!(1592)!0.096%
0.011!H526Y%
Rifampicin!
rpoB%CAC!:!TAC!(1376)!
0.073%0.014!
H526D%Rifam
picin!rpoB%
CAC!:!GAC!(1576)!0.064%
0.017!!
Table S2. Genotypes of the evolved clones from competition between H526YRif and S531FRif
!!
!!
Genotype(of(H526Y(Evolved(ClonesgalK:YFP,*rpoB*(H526Y),*rpoA*(T196I),*waaZ*(S280P)galK:YFP,*rpoB*(H526Y),*rpoA*(T196I)galK:YFP,*rpoB*(H526Y),*rpoA*(T196I)galK:YFP,*rpoB*(H526Y),*rpoA*(T196I)galK:YFP,*rpoB*(H526Y),*rpoA*(T196I)galK:YFP,*rpoB*(H526Y),*rpoA*(T196I)galK:YFP,*rpoB*(H526Y),*rpoA*(T196I)galK:YFP,*rpoB*(H526Y),*rpoA*(T196I)galK:CFP,*rpoB*(H526Y),*rpoC*(Unknown)galK:CFP,*rpoB*(H526Y),*yifE*(G39D)galK:CFP,*rpoB*(H526Y),*rpoC*(R1224C)galK:CFP,*rpoB*(H526Y),*rpoC*(S486P)galK:CFP,*rpoB*(H526Y),*rpoC*(S486P)galK:CFP,*rpoB*(H526Y),*rpoC*(S486P)galK:CFP,*rpoB*(H526Y),*rpoC*(H540P)
Genotype(of(S531F(Evolved(ClonesgalK:YFP,*rpoB*(S531F),*envR*(IS5)galK:YFP,*rpoB*(S531F),*znuA*(F37L),*sspA*(L156P)galK:YFP,*rpoB*(S531F),*znuA*(F37L),*sspA*(L156P)galK:YFP,*rpoB*(S531F),*yeaR*(IS186)galK:YFP,*rpoB*(S531F),*nadK/recN*(Intergenic),*yedW*(IS1)galK:YFP,*rpoB*(S531F),*rpoC*(K50T)galK:YFP,*rpoB*(S531F),*paoB*(V231E)galK:YFP,*rpoB*(S531F),*nrfG/gltP*(Intergenic)