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.

Ê

Ê

ÊÊ

Ê

ÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊ

ÊÊÊÊÊÊÊÊÊ

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

ÊÊ

Ê

Ê

ÊÊ

ÊÊÊ

Ê

ÊÊÊÊ

ÊÊÊÊÊ

ÊÊ

Ê

Ê

ÊÊ

ÊÊÊ

Ê

ÊÊÊÊ

ÊÊÊÊÊ

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)