thrl leuo/leul degp lacz ybao [MarA]

Transcription

1 a thrl leuo/leul degp lacz yao [MarA] complex free DNA phep mode/acrz yiv yjc/grxa ycgf/ycgz [MarA] complex free DNA fnr yneo marc/marr yeef ompc/micf [MarA] complex free DNA ypec yfes><cysm guab/xsea ETEC_32 nudf/tolc [MarA] complex free DNA

2 yhv mlaf/yrg ipa/yidq mnmg yiht [MarA] complex free DNA yiig ETEC_434 ETEC_437 yjcb/yjcc yjjp/yjjq [MarA] complex free DNA ETEC_472 deob p948_77 [MarA] complex free DNA cydd yis/t ETEC 2117 cyoa PestA1 [MarA] free DNA Supplementary Figure 1: Binding of MarA to DNA fragments derived from ChIP-seq inding peaks for MarA. Panel a) illustrates results of electrophoretic moility shift assays with different DNA fragments containing the marox. Panel shows inding of equivalent MarA concentrations to DNA fragments containing no marox. MarA was added at a final concentration of.3, 1., and 1.7 mm as indicated y the triangle. The location of free DNA, and DNA ound y MarA, is indicated.

3 CIPROFLOXACIN-.4 CIPROFLOXACIN-.6 CIPROFLOXACIN-.8 NALIDIXICACID-.5 NALIDIXICACID-1. NALIDIXICACID-1.5 NALIDIXICACID-2. NORFLOXACIN-.1 NORFLOXACIN-.2 NORFLOXACIN-.4 LEVOFLOXACIN-.2 NOVOBIOCIN-1 NOVOBIOCIN-12 NOVOBIOCIN-3 NOVOBIOCIN-4 NOVOBIOCIN-6 NOVOBIOCIN-8 SULFAMETHIZOLE-1 SULFAMETHIZOLE-2 SULFAMETHIZOLE-3 SULFAMONOMETHOXINE-1 SULFAMONOMETHOXINE-5 TRIMETHOPRIM-.1,SULFAMETHIZOLE-5 TRIMETHOPRIM-.1 TRIMETHOPRIM-.2 TRIMETHOPRIM-.3 TRIMETHOPRIM-.4 ERYTHROMYCIN-.1 ERYTHROMYCIN-1. ERYTHROMYCIN-1. ERYTHROMYCIN-5. CLARYTHROMYCIN-.1 CLARYTHROMYCIN-1. CLARYTHROMYCIN-1. CLARYTHROMYCIN-5. SPIRAMYCIN-1 SPIRAMYCIN-2 SPIRAMYCIN-5 AZITHROMYCIN-.2 AZITHROMYCIN-.1 AZITHROMYCIN-1. CHLORAMPHENICOL-.5 CHLORAMPHENICOL-1. CHLORAMPHENICOL-1.5 CHLORAMPHENICOL-2. FUSIDICACID-1 FUSIDICACID-2 FUSIDICACID-5 FUSIDICACID-5 GENTAMICIN-.5 GENTAMICIN-.1 STREPTOMYCIN-.5 AMIKACIN-.5 AMIKACIN-.1 AMIKACIN-.2 SPECTINOMYCIN-4. SPECTINOMYCIN-6. PUROMYCIN-1 PUROMYCIN-25 PUROMYCIN-5 TOBRAMYCIN-.5 TOBRAMYCIN-.1 TOBRAMYCIN-.2 TOBRAMYCIN-.4 DOXYCYCLINE-.25 DOXYCYCLINE-.5 DOXYCYCLINE-.75 DOXYCYCLINE-1. MINOCYCLINE-.2 MINOCYCLINE-.5 MINOCYCLINE-1. TETRACYCLINE-.25 TETRACYCLINE-.5 TETRACYCLINE-.75 TETRACYCLINE-1. AZTREONAM-.2 AZTREONAM-.4 CARBENICILLIN-.5 CARBENICILLIN-1. CARBENICILLIN-1.5 AMOXICILLIN-.25 AMOXICILLIN-.5 AMOXICILLIN-1. AMOXICILLIN-1.5 AMPICILLIN-1. AMPICILLIN-2. AMPICILLIN-4. AMPICILLIN-8. MECILLINAM-.3 MECILLINAM-.6 MECILLINAM-.9 MECILLINAM-.12 OXACILLIN-.5 OXACILLIN-4. OXACILLIN-5. CEFTAZIDIME-.25 CEFTAZIDIME-.5 CEFTAZIDIME-.75 CEFSULODIN-6.,MECILLINAM-.3 CEFACLOR-1. CEFACLOR-2. CEFACLOR-3. CEFSULODIN-12. CEFSULODIN-18. CEFSULODIN-24. CEFSULODIN-6. CEFOXITIN-.25 CEFOXITIN-.5 CEFOXITIN-.75 CEFOXITIN-1. CHIR9-.2 CHIR9-.25 CHIR9-.4 CHIR9-.5 CHIR9-.75 ISONIAZID-.2 ISONIAZID-1. ISONIAZID-1.5 VANCOMYCIN-1 VANCOMYCIN-2 VANCOMYCIN-5 FOSFOMYCIN-1. GLUFOSFOMYCIN-.5 GLUFOSFOMYCIN-.2 POLYMYXINB-1. POLYMYXINB-2. POLYMYXINB-4. POLYMYXINB-6. BACITRACIN-1 BACITRACIN-2 BACITRACIN-3 A A A A INDOLICIDIN-.1 MMS-.5% NIGERICIN-.1 NIGERICIN-1. NIGERICIN-5. BICYCLOMYCIN-1 BICYCLOMYCIN-1 RIFAMPICIN-1. RIFAMPICIN-2. TUNICAMYCIN-1. TUNICAMYCIN-3. TUNICAMYCIN-7.5 THIOLACTOMYCIN-1 THIOLACTOMYCIN-5 THIOLACTOMYCIN-5 STREPTONIGRIN-.1 STREPTONIGRIN-.2 STREPTONIGRIN-.5 NITROFURANTOIN-.1 NITROFURANTOIN-.5 NITROFURANTOIN-1. NITROFURANTOIN-1.5 MarA targets degp deob fnr grxa guab IpA leuo marr micf mnmg mode nudf ompc phep thra tolc xsea yao yiv yjc ycgf ycgz yeef yidq yjcb yjcc yjjp yjjq ypec mlaf yrg Drug treatment Supplementary Figure 2. Phenotypic landscape of the MarA regulon. The heatmap illustrates fitness scores 29 of strains lacking MarA target genes (y-axis) compared to the wild type parent strain. Red indicates a fitness decrease and pale lue indicates a fitness increase. Strains were grown in the presence of different antiiotics (x-axis). The antiiotics are clustered according to the cellular process targeted. Individual row and column names are provided. See reference 29 for further details.

4 Miller units marox - marox The f from The f IPTG prw mlaf [IPTG] mlafedcb xsea Supplementary Figure 3: Activation of mlafedcb and xsea y increasing intracellular MarA: a requirement for the marox. The result of a -galactosidase assay done using lysates of T7 express cells transformed with derivatives of the lacz reporter plasmid, prw5. Activity values otained using empty prw5 vector have een sutracted. Additional MarA is provided y plasmid pet21amara that encodes mara under the control of an IPTG inducile promoter. Error ars represent standard deviation (n=3).

5 OD 65 OD no plasmid pbr322 pbr322 xsea - ciprofloxacin + ciprofloxacin wild type xsea::kan - ciprofloxacin + ciprofloxacin - ciprofloxacin no plasmid pbr322 pbr322 xsea + ciprofloxacin minutes Supplementary Figure 4: Complementation of the xsea::kan phenotype y xsea. The graph shows OD 65 values otained for liquid cultures of strain BW25113 xsea::kan grown in the presence or asence of.5 g/ml ciprofloxacin. The BW25113 xsea::kan cells were transformed with pbr322 derivatives encoding xsea under the control of the xsea1 fragment. Error ars represent standard deviation (n=3).

6 a XseA domain organisation domain oundaries OB fold catalytic domain coiled coils CTD OD mutations F63A R25A Q267Stop P353Stop pbr322 xsea pbr322 xsea F63A pbr322 xsea R25A - ciprofloxacin - ciprofloxacin - ciprofloxacin OD ciprofloxacin + ciprofloxacin + ciprofloxacin OD 65 OD pbr322 xsea Q267Stop - ciprofloxacin + ciprofloxacin minutes minutes pbr322 xsea P353Stop - ciprofloxacin + ciprofloxacin Supplementary Figure 5: Complementation of the xsea::kan phenotype requires XseA determinants for interaction with XseB. Panel a) shows a graphical illustration of XseA domain organisation. Mutations introduced into pbr322 encoded xsea are shown elow that schematic. In panel ) the graphs show OD 65 values for liquid cultures of strain BW25113 xsea::kan grown in the presence or asence of.5 g/ml ciprofloxacin. The BW25113 xsea::kan cells were transformed with pbr322 derivatives encoding xsea under the control of the xsea1 fragment. Error ars represent standard deviation (n=3).

7 Miller units a mlaf1.1 TTTATGCGGCTAAAAAGTAAAACAAATGCCAGCTTTCGCTAACCACGGCGGGTAAGGTTCTGTAAA marox P2mlaF P3mlaF TATGTTGGGTTCAAGGTTAAATTGAGCGCCATGCTTAGAAAATCAACGCAAGACGAAGGGTGAATTATG mlaf2.1 TTTATGCGGCTAAAAAGTAAAACAAA TGCCAGCTTTCGCTACCACGGCGGGTAAGGTTCTGTAAA P2mlaF P3mlaF TATGTTGGGTTCAAGGTTAAATTGAGCGCCATGCTTAGAAAATCAACGCAAGACGAAGGGTGAATTATG marox - marox mlaf1.1 mlaf2.1 mlafedcb promoter derivative Supplementary Figure 6: Effect of mlafedcb P1 promoter inactivation. Panel a) shows the DNA sequence of the mlaf1.1 and mlaf2.1 DNA fragments. The mlaf start codon is shown in lue and the marox is in green. Transcription start sites (+1) are underlined and further highlighted y a ent arrow. The P1 promoter has een inactivated y changing the sequence of the -1 element from 5'-TATTCT-3' to 5'-GGTTCT-3. The associated mutations are highlighted y the red ox. Panel ) shows results of -galactosidase assays using lysates of strain JCB387 transformed with prw5 carrying either the mlaf1.1 or mlaf2.1 fragment upstream of lacz. Error ars represent standard deviation (n=3).

8 OD 65 OD no plasmid pbr322 pbr322 mlafedcb - doxycycline - doxycycline - doxycycline no plasmid pbr322 pbr322 mlafedcb + doxycycline + doxycycline + doxycycline minutes Supplementary Figure 7: Complementation of the mlae::kan phenotype y mlafedcb. The graph shows OD 65 values otained for liquid cultures of strain BW25113 mlae::kan grown in the presence or asence of 1. g/ml doxycycline. The BW25113 mlae::kan cells were transformed with pbr322 derivatives encoding mlafedcb under the control of the mlaf1 fragment. Error ars represent standard deviation (n=3).

9 Relative crystal violet inding (aritrary units) Doxycycline accumulation (aritrary units) % asorance of aqueous phase a marox - marox 1 4 c 2 1 Time (minutes) + marox - marox E. coli strain + marox - marox volume of xylene (ml) Supplementary Figure 8: The mlafedcb marox is required for effective arrier function and optimal surface hydrophoicity. Panel a) shows accumulation of doxycycline as a function of time for BW25113 mlae::kan cells transformed with pbr322 encoding mlafedcb under the control of the mlaf1 (+marox, solid line) or the mlaf2 (-marox, dashed line) promoter fragments. Panel ) depicts changes in asorance of an aqueous suspension of acterial cells after mixing with p-xylene. The % asorance is relative to that otrained with no p-xylene, at equilirium. Data points are coloured as in panel a). The indicated volume of p-xylene is shown on the x-axis. The ar graph (c) shows relative crystal violet adsorption y BW25113 mlae::kan cells transformed with pbr322 encoding mlafedcb under the control of the mlaf1 (solid ar) or the mlaf2 (open ar). Error ars represent standard deviation (n=3).

10 complexes complexes a MarA Ro SoxS - Lane Free DNA PestA1 (control DNA) SoxS MarA Ro SoxS - Lane l DNA) marrab Free DNA Supplementary Figure 9: Ro inds DNA with high affinity ut low specificity. Panel a) shows the results of an electrophoretic moility shift assay. Proteins (.4, 1.2 or 2 M) were incuated with the PestA1 DNA fragment that does not contain a marox. Panel ) shows inding of MarA and SoxS (.4, 1.2 or 2 M) or Ro (.8,.24 or.4 M) to a DNA fragment containing the marrab promoter.

11 complexes complexes % ound % ound DNA DNA complexes complexes % ound % ound DNA DNA a a - - MarA Ro SoxS MarA Ro SoxS Lane xsea xsea Free DNA Free DNA MarA inding SoxS MarA inding SoxS inding [protein].8 M.24.4 [protein] M mlafedcb MarA mlafedcb Ro SoxS MarA Ro SoxS MarA Ro SoxS MarA - - Ro SoxS Lane MarA inding SoxS MarA inding SoxS inding mlafedcb mlafedcb Free DNA Free DNA [protein] M [protein] M Supplementary Figure 1: The xsea and mlafedcb and regulatory regions preferentially ind MarA rather than SoxS. The figure shows inding of MarA, Ro or SoxS to the a) xsea1 or ) mlaf1 DNA fragments. For each experiment, the % free DNA was determined for each protein concentration as illustrated in the line graphs.

12 a Figure 2c top Figure 2c ottom Figure 2d Figure 2h

13 Figure 3f Figure 3c top Figure 3c ottom Figure 3d

14 c thrl leuo/leul lacz yao phep ycgf/ycgz marc/marr yjc/grxa ypec fnr ETEC_2117

15 guab/xsea mlaf/yrg yidq/ipa mnmg yjjp/yjjq cydd yis/yit cyoa

16 yiig ETEC_434 ETEC_437 yjcb ETEC_472 deob yiht tolc p948_77 degp yeef micf yfes

17 yhv ETEC_32 acrz yneo yiv PestA1 d Supplementary Fig. 9a Supplementary Fig. 9 Supplementary Fig. 1a Supplementary Figure 11: Uncropped gel images. The images correspond to gels presented in a) Figure 2 ) Figure 3 c) Supplementary Fig. 1 and d) Supplementary Fig. S9-1.