Potential for adaptation overrides cost of resistance

(1)

VS H526Y x8 C re ate p op ula tio ns po lymo rp hic fo r an tib io tic re si st an ce mu ta tio n. Pro pa ga te fo r 30 d ays in rich , d ru g f re e, me dia . Bo ttle ne ck eve ry 24 h. An alyse a nd fit ma th ema tica l mo de l to d yn ami cs of mo re co st ly re si st an ce . Ê Ê Ê Ê Ê Ê Ê Ê Ê Ê Ê Ê Ê Ê Ê Ê Ê Ê Ê Ê Ê Ê Ê Ê Ê Ê Ê Ê Ê Ê Ê

24h

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ig

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1

H526D H526Y H526D x8 VS H526Y x8 S531F H526Y S531F x8 VS x8 S531F S531F x8 K 43T K 43T

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(2)

y = -0 .0 74 4x - 0.1 55 7 -1 0 -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

B

C

F

ig

ure

2

y = -0 .0 74 4x - 0.1 55 7 -1 0 -8 -6 -4 -2 0 2 0 50 100 150 200 250 300

Ln(H526Y/H526D)

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(3)

y = -0 .0 15 x - 0.5 87 5 -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

B

C

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(4)

y = -0 .0 31 9x - 0.6 43 8 -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

B

C

F

ig

ure

4

y = -0 .0 31 9x - 0.6 43 8 -7 -5 -3 -1 ₁ ₃ ₅ ₇ ₉ 11 0 50 100 150 200 250 300

Ln(K43T/S531F)

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(5)

-0 .0 9 -0 .0 4 0.01 0.06 0.1 1 0.16 rp oA_(T 19 6I) rp oC (R 12 24 C) rp oA (T 19 6I),_w aa Z (S2 80_P) yifE (G 39 D) rp oC (S4 86_P) rp oC (H 45 0P) Selective Effect vs O w n Ba ckg ro un d (H 52 6Y) vs C omp eti to r Ba ckg ro un d (S5 31 F )

A

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531F

-0 .0 9 -0 .0 4 0.01 0.06 0.1 1 0.16 rpoC_(K5 0T) nrfG /gltP nadK/ recN , ye dW (IS1 ) znuA_(F3 7L),_ssp A (L 156P) paoB _(V2 31E) yeaR_(IS1 86) envR (IS5 ) Selective Effect vs O w n Ba ckg ro un d (S5 31 F ) vs C omp eti to r Ba ckg ro un d (H 52 6Y)

(6)

Mutation in Resistance 2 Backgr ound (N R2 ) Fitness W NR2 = 1.027 Time T NR2 = 3

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6 Experimental

Inferr

ed

Time

A

B

LN

Resistance 2 Resistance 1

(

)

LN

Resistance 2 Resistance 1

(

)

W R1 =1 W R2 =0.96 Mutation in Resistance 2 Backgr ound (N R2 ) Fitness W NR2 = 1.057 Time T NR2 = 3 Mutation in Resistance 1 Backgr ound (N R1 ) Fitness W NR1 = 1.068 Time T NR1 = 29

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6 Experimental

Inferr

ed

Ê Ê Ê Ê Ê Ê Ê Ê Ê Ê Ê Ê Ê Ê Ê Ê Ê Ê Ê Ê Ê Ê Ê Ê Ê Ê Ê Ê Ê Ê Ê Ê Ê Ê Ê Ê Ê Ê W R1 =1 W R2 =0.96

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(7)

y = -0.015x - 0.5875 -3 -2 -1 0 1 2 3 4 5 6 0 50 100 150 200 250 300 L n (H 52 6Y/ S5 31 F ) y = -0.0744x - 0.1557 -10 -8 -6 -4 -2 0 0 50 100 150 200 250 300 L n (H 52 6Y/ H 52 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 L n (K 43 T /S5 31 F )

(8)

−7 −5 −3 −1 0 1 3 5 7 9 11 0 25 50 75 100 125 150 175 200 225 250 275 Generations LN(K43T/S531F) −3 −2 −1 0 1 2 3 4 5 6 0 25 50 75 100 125 150 175 200 225 250 275 Generations LN(H526Y/S531F) −10 −8 −6 −4 −2 0 2 0 25 50 75 100 125 150 175 200 225 250 275 Generations LN(H526Y/H526D)

A

B

C

(9)

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(10)

H 52 6D &(R1 ) H 52 6Y &(R2 ) Ini/ al& H526Y& Fr eq ue nc y W& T W& T Po pu la/ on &1 0. 538 1. 576 11 1. 552 15 Po pu la/ on &2 0. 382 1. 293 11 1. 263 22 Po pu la/ on &3 0. 488 1. 387 41 1. 348 51 Po pu la/ on &4 0. 468 1. 483 26 1. 44 32 Po pu la/ on &5 0. 413 1. 421 21 1. 409 30 Po pu la/ on &6 0. 476 1. 474 31 1. 478 42 Po pu la/ on &7 0. 513 1. 218 11 1. 19 19 Po pu la/ on &8 0. 493 1. 776 18 1. 767 23 Po pu la/ on &9 0. 557 1. 017 3 Po pu la/ on &1 0 0. 461 1. 028 29 Po pu la/ on &1 1 0. 581 1. 023 3 Po pu la/ on &1 2 0. 58 1. 002 46 1. 02 3 Po pu la/ on &1 3 0. 523 1. 023 14 Po pu la/ on &1 4 0. 55 1. 001 38 1. 028 23 Po pu la/ on &1 5 0. 59 1. 668 37 1. 671 45 T ab le 1. Evol u ti on ar y P ar ame te rs e sti mate d for th e comp eti ti on b etw ee n s tr ai n s H 526Y an d H 526D . W sta nds for the fi tne ss of the e m ergi ng ha pl ot ype a nd T it s t im e of appe ara nc e. Ini tia l F re q sta nds for the infe rre d ini tia l fre que nc y of the H 526Y ba ckground. In the c as es w he re onl y one of the ba ckgrounds ha s ac qui re d a m ut ati on, t he infe rre d pa ra m ete rs a re in bol d for t he ba ckground w he re no m ut ati on w as infe rre d.

(11)

S5 31 F& (R1 ) H 52 6Y &(R2 ) Ini1 al& H526Y& Fr eq ue nc y W& T W& T pu la1 on &1 0. 369 1. 459 15 1. 472 17 pu la1 on &2 0. 458 1. 041 46 1. 051 3 pu la1 on &3 0. 277 1. 436 15 1. 45 19 pu la1 on &4 0. 472 1. 034 7 1. 054 4 pu la1 on &5 0. 286 1. 529 8 1. 554 12 pu la1 on &6 0. 454 1. 585 55 1. 637 60 pu la1 on &7 0. 381 1. 615 36 1. 655 40 pu la1 on &8 0. 325 1. 465 11 1. 491 15 pu la1 on &9 0. 473 1. 199 19 1. 194 14 pu la1 on &1 0 0. 378 1. 163 14 1. 154 10 pu la1 on &1 1 0. 356 1. 595 17 1. 601 19 pu la1 on &1 2 0. 483 1. 028 3 1. 049 4 pu la1 on &1 3 0. 37 1. 344 11 1. 349 12 pu la1 on &1 4 0. 444 1. 542 9 1. 562 12 pu la1 on &1 5 0. 446 1. 505 16 1. 514 17 pu la1 on &1 6 0. 468 1. 647 55 1. 7 59 T ab le 2. Evol u ti on ar y P ar ame te rs e sti mate d for th e comp eti ti on b etw ee n s tr ai n s H 526Y an d S 531F . T he m ea ni ng of t he pa ra m ete rs is a s i n Ta bl e 1.

(12)

T ab le 3. Evol u ti on ar y P ar ame te rs e sti mate d for th e comp eti ti on b etw ee n s tr ai n s K43T an d S 531F . T he m ea ni ng of t he pa ra m ete rs is a s i n Ta bl e 1. S5 31 F& (R1 ) K4 3T &(R2 ) Ini1 al& K43T & Fr eq ue nc y W& T W& T Po pu la1 on &1 0. 505 1. 207 16 1. 188 3 Po pu la1 on &2 0. 319 1. 349 20 1. 359 26 Po pu la1 on &3 0. 299 1. 546 18 1. 547 21 Po pu la1 on &4 0. 349 1. 26 16 1. 253 18 Po pu la1 on &5 0. 319 1. 669 4 1. 737 5 Po pu la1 on &6 0. 359 1. 457 4 1. 474 10 Po pu la1 on &7 0. 333 1. 088 52 1. 06 34 Po pu la1 on &8 0. 256 1. 325 7 1. 334 12 Po pu la1 on &9 0. 43 1. 687 37 1. 732 42 Po pu la1 on &1 0 0. 335 1. 097 3 1. 09 4 Po pu la1 on &1 1 0. 357 1. 537 50 1. 529 53 Po pu la1 on &1 2 0. 388 1. 16 11 1. 144 6 Po pu la1 on &1 3 0. 41 1. 195 5 1. 193 7 Po pu la1 on &1 4 0. 379 1. 095 43 1. 058 6 Po pu la1 on &1 5 0. 329 1. 663 25 1. 672 30

(13)

bl e 4 . Po te n tia l c o m p e n s a to ry m ut a tions ide nt ifi e d in the ge nom e s of the cl o n es ev o lv ed in th e co m p et iti o n b et w een r esi st an ces 2 6 Y a n d S 5 3 1 F . G en o m e Po siti o n G en e(s ) Mu ta ti o n A n n o ta ti o n Frequency F u n cti o n 3,438,465 rp oA C → T T196I 51.5% R N A p olyme ra se , a lp ha su bu nit 4,184,828 rp oC T → C S4 86 P 25.5% 4,187,042 rp oC C → T R1224C 6.1% 4,184,721 rp oC A → C H450P 2.9% 3,797,382 waaZ T → C S2 80 P 19.7% Lip op olysa cch ari de co re b io syn th esi s pro te in 3,946,224 yi fE G → A G39D 12.0% C on se rve d p ro te in , U PF 04 38 fa mi ly, u nkn ow n f un ct io n 4,292,389 nrf G /g ltP A → T In te rg en ic 5.0% [n rfG ] H eme lya se (N rf EF G ) fo r in se rt io n o f h eme in to c5 52 , su bu nit N rf G /[g ltP ] g lu ta ma te /a sp art ate : p ro to n symp ort e 3,637,091 pitA C → T A4 76 V 2.6% Ph osp ha te tra nsp ort er, lo w ‑ aff in ity; te llu rite imp ort er 374,196 mh pE/ mh pT T → C In te rg en ic 2.4% 4 ‑ hyro xy ‑ 2 ‑ oxo va le ra te /4 ‑ hyd ro xy ‑ 2 ‑ oxo pe nta no ic aci d a ld ola se , cl ass I/p uta tive 3 ‑ hyd ro xyp he nyl pro pio nic tra nsp ort er 3,464,375 bfr C → T R125C 2.0% Ba ct eri ofe rri tin , iro n st ora ge a nd d eto xi fica tio n p ro te in 1,743,180 yd hQ T → C S3 24 S 3.1% H yp oth eti ca l p ro te in 3,898,043 yi eL C → A T186T 2.5% Pu ta tive xyl an ase 4,183,521 rp oC A → C K5 0T 14.8% R N A p olyme ra se , b eta p rime su bu nit 4,292,389 nrf G /g ltP A → T In te rg en ic 19.6% [n rfG ] H eme lya se (N rf EF G ) fo r in se rt io n o f h eme in to c5 52 , su bu nit N rf G / [g ltP ] G lu ta ma te /a sp art ate :p ro to n symp ort e 2,749,808 na dK/ re cN Δ 1 bp In te rg en ic 18.1% [n ad K ] N AD ki na se / [ recN ] R eco mb in ati on a nd re pa ir pro te in 1,940,499 zn uA T → C F37L 17.7% Z in c tra nsp ort er su bu nit : p eri pla smi c ‑ bin din g co mp on en t o f ABC su pe rf ami ly 3,374,976 ssp A T → C L156P 12.2% St rin ge nt st arva tio n p ro te in A 300,420 pa oB T → A V2 31 E 11 .8 % Pa oABC a ld eh yd e o xi do re du ct ase , F AD ‑ co nta in in g su bu nit 2,036,728 ye dW IS1 In s Coding 9.9% Pu ta tive D N A ‑ bin din g re sp on se re gu la to r in tw o ‑ co mp on en t syst em w ith Ye dV 1,877,853 ye aR IS1 86 In s Coding 8.4% H yp oth eti ca l p ro te in 3,410,893 en vR IS5 In s Coding 6.8% DNA ‑ bin din g t ra nscri pti on al re gu la to r 2,361,326 yf aY A → G V1 10 V 9.4% H yp oth eti ca l p ro te in S5 31 F R N A p olyme ra se , b eta p rime su bu nit H526Y !

(14)

Tab le S 1. F itne ss c os ts i m pos ed by t he a nt ibi ot ic re sis ta nc e a lle le s ( K43T , S531F , H 526Y , H 526D ) m ea sure d i n c om pe tit ion a ga ins t t he s ens iti ve re fe re nc e s tra in. ! ! ! !!

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K43T

%

Str ep to m yc in ! rpsL % AAA! :!AC A! (128) ! 0. 092 % 0. 034 !

S531F

%

Ri fam pic in ! rpoB % TCC! :!TTC!(1 59 2)! 0. 096 % 0. 011 !

H526Y

%

Ri fam pic in ! rpoB % CA C! :!TA C!(1 37 6)! 0. 073 % 0. 014 !

H526D

%

Ri fam pic in ! rpoB % CA C! :!GAC!(1576) ! 0. 064 % 0. 017 ! !

(15)

and S531FRif ! ! ! ! Genotype(of(H526Y(Evolved(Clones galK: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(Clones galK: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)