Supporting Information

Lee et al. 10.1073/pnas.0903619106 SI Materials and Methods The tobramycin sensitivities for strains expressing the rmtD Plasmids. Plasmid p(amgRSϩ) was constructed by inserting a 2.4 ribosomal methylase gene were determined by a 2-fold broth kb PCR fragment carrying the region from 206 bp upstream of dilution test. Inocula were prepared by growing cells at 37 °C for the amgR start codon to 60 bp downstream of the amgS 16 h in LB lacking NaCl. Cells were diluted to approximately ϫ 6 termination codon into a derivative of pUCP19 (1). Plasmid 3.3 10 cells/ml based on OD600. After a 90 min incubation at 37 °C, 10 ␮l aliquots were distributed to wells (in 96-well format) pRmtD was constructed by inserting the 2.3 kb HindIII-XbaI ␮ fragment carrying rmtD from pPA95B1 (2) into a derivative of containing 125 l LB buffered with 0.1 M MOPS at pH 7.6 pUCP19 (1). Plasmids used for complementation studies (Table containing different concentrations of tobramycin. MICs were 1) were constructed in pUCP19 derivatives by inserting PCR determined after incubation for 24 h at 37 °C. fragments corresponding to the genes tested. Plasmid pIbsC was Transcriptional Profiling. MPAO1 and ⌬amgRS transcriptomes constructed by inserting a 1.8 kb PCR fragment carrying the were compared under tobramycin-treated and -untreated con- arabinose-inducible ibsC construct (P -ibsC) from pAZ3-ibsC bad ditions. Samples were prepared in duplicate from independent (3) into a derivative of pUCP19 (4), resulting in pIbsC. The cultures grown in 15 ml LB lacking NaCl. Cultures were incu- plasmid carrying mexEF and oprN used for complementation has bated at 37 °C with shaking (250 rpm) and harvested at OD600 been described (5). 1.0. Treated cultures received 1 ␮g/ml tobramycin 15 min before harvesting. Approximately 2 ϫ 109 cells were mixed with Antibiotic Sensitivity Assays. In general, strains to be assayed for RNAprotect Bacteria reagent (Qiagen). RNA purification and antibiotic sensitivity were grown overnight at 37 °C in LB in cDNA synthesis, purification, and fragmentation were per- 96-well format, then diluted 100-fold and incubated 90 min at formed as previously described (6). cDNA was labeled with ␮ 5 37 °C. Aliquots (8 l; approximately 10 cells) were spotted on GeneChip DNA Labeling reagent (Affymetrix). Hybridization LB agar containing antibiotic. Test plates were incubated for to Affymetrix PAO1 GeneChips was carried out at the Univer- 18 h and 42 h at 37 °C and examined for growth of the spots. The sity of Washington Center for Array Technologies. Expression MIC was defined as the lowest antibiotic concentration prevent- data were normalized using Affymetrix GeneChip Operating ing visible lawn growth. In some assays, NaCl was omitted from Software Version 1.4 and gene expression differences between the medium, or the medium was buffered using 0.1 M MOPS (pH the tobramycin-treated wild-type and ⌬amgRS cultures were 7.6), 0.1 M MES (pH 5.5 or 6.0) or 0.1 M TAPS (pH 8.5). For determined using Cyber-T (http://cybert.microarray.ic- strains carrying pIbsC, media were supplemented with 150 ␮g/ml s.uci.edu/). Genes exhibiting a significance threshold of P Ͻ carbenicillin (for plasmid maintenance) and 0.025% arabinose 0.001 were deemed differentially expressed, resulting in a pos- (to induce IbsC). terior probability of differential expression greater than 0.97.

1. Schweizer HP (1992) Allelic exchange in Pseudomonas aeruginosa using novel ColE1- 5. Maseda H, Yoneyama H, Nakae T (2000) Assignment of the -selective subunits type vectors and a family of cassettes containing a portable oriT and the counter- of the MexEF-OprN multidrug efflux pump of Pseudomonas aeruginosa. Antimicrob selectable Bacillus subtilis sacB marker. Mol Microbiol 6:1195–1204. Agents Chemother 44(3):658–664. 2. Doi Y, de Oliveira Garcia D, Adams J, Paterson DL (2007) Coproduction of novel 16S 6. Schuster M, Lostroh CP, Ogi T, Greenberg EP (2003) Identification, timing, and signal rRNA methylase RmtD and metallo-beta-lactamase SPM-1 in a panresistant Pseudo- specificity of Pseudomonas aeruginosa quorum-controlled genes: A transcriptome monas aeruginosa isolate from Brazil. Antimicrob Agents Chemother 51:852–856. analysis. J Bacteriol 185:2066–2079. 3. Fozo EM, et al. (2008) Repression of small toxic protein synthesis by the Sib and OhsC small RNAs. Mol Microbiol 70:1076–1093. 4. West SE, Schweizer HP, Dall C, Sample AK, Runyen-Janecky LJ (1994) Construction of improved Escherichia-Pseudomonas shuttle vectors derived from pUC18/19 and se- quence of the region required for their replication in Pseudomonas aeruginosa. Gene 148:81–86.

Lee et al. www.pnas.org/cgi/content/short/0903619106 1of15 vrta bevdfrpamdfe PO,sgetn hta nrae ee ftergltricessitiscrssac oteatboi.wt antibiotic. the to resistance intrinsic increases regulator the of level increased an that suggesting MPAO1, plasmid-free ϩ for observed that over e tal. et Lee p( plasmid containing or lacking strains in presented are S1. Fig. ,p( amgRS www.pnas.org/cgi/content/short/0903619106 opeetto of Complementation ϩ ); ⌬ , ⌬ amgRS ;R,

amgR MIC (µg/ml) 0.25 amgRS 1.0 4.0 ::IS uat.Tetbayi estvte fwl-ye(MPAO1), wild-type of sensitivities tobramycin The mutants. phoA hhM;S, M7; /hah wt amgRS amgS wt/+ ::IS ϩ .Tepeec ftepamdi h ifrn tan lvtdtbayi eitne2 o4-fold to 2- resistance tobramycin elevated strains different the in plasmid the of presence The ). lacZ hhA3 /hah Genotype /+ R ⌬ amgRS / S/+ S R/+ and , amgR and amgS rnpsnisrinmutants insertion transposon MPAO1; , 2of15 amgR e tal. et Lee IS PA3303 a carrying strain IS, Control parent; MPAO1, 8-fold. function the carrying resistance strains the of determinations MIC tobramycin duplicate S2. Fig. Control IS/pRmtD amgR MPAO1 /pRmtD ::IS, amgRS www.pnas.org/cgi/content/short/0903619106 amgR nacdtbayi estvt of sensitivity tobramycin Enhanced ::IS /pRmtD ::IS phoA /pRmtD hhM7. /hah { { { {

amgRS 0 uatdrvtvso tanepesn 6 RAmtyaerssac ucin h mg shows image The function. resistance methylase rRNA 16S a expressing strain a of derivatives mutant 2 rmtD

4 Tobramycin [

ee(npamdpmD.The pRmtD). plasmid (in gene 8 16

phoA 32 hhisrinwihde o hneaiolcsd sensitivity; aminoglycoside change not does which insertion /hah µ

64 g/mL] ⌬ amgRS 128

uainrdcdteMCo tan expressing strains of MIC the reduced mutation 256 512 1024 2048 [ µ MIC 512 512 g/mL] 64 64 3of15 Fig. S3. High pH-sensitive growth of amgRS mutants. The figure shows spots of 10-fold dilutions of strains grown overnight at 37 °C on LB agar buffered at pH 5.5 or 8.5. The amgR::IS and ⌬amgRS mutants exhibit reduced growth and plating efficiency at pH 8.5, whereas parent strains, the complemented ⌬amgRS mutant and a mexY tobramycin-sensitive strain plated normally. All strains grew well at pH 5.5, forming colonies of comparable sizes. The ⌬amgRS mutant maintained wild-type plating efficiency and colony size at pH Յ7.25. Wild-type, MPAO1; IS control, MPAO1 carrying an insertion in PA3303 with no apparent effect on aminoglycoside sensitivity; mexYϪ, mexY::ISlacZ/hah B19; amgRϪ, amgR::ISphoA/hah M7; p(amgRSϩ), plasmid carrying wild-type amgRS genes.

Lee et al. www.pnas.org/cgi/content/short/0903619106 4of15 rwhatr4 sson pIbsC: shown. is h 48 after Growth e tal. et Lee of Induction shown. are tobramycin of S4. Fig. MPAO1 amgRS www.pnas.org/cgi/content/short/0903619106 nacdtbayi estvt of sensitivity tobramycin Enhanced pIbsC + + + + Arabinose ϩ Induction lsi arigP carrying plasmid , ibsC yaaioeadto eutdi n8fl euto ntbayi I o PO n the and MPAO1 for MIC tobramycin in reduction 8-fold an in resulted addition arabinose by + + + + .aeruginosa P. bad xrsigthe expressing ibsC 0 ; Ϫ mt etr rbns induction: Arabinose vector. empty , 0.004 ibsC Tobramycin [ eefrom gene 0.008

.coli E. 0.016 rwhof Growth . 0.031 µ ϩ g/mL] .2%arabinose; 0.025% , .aeruginosa P. 0.063

0.125 nL grcnann ifrn levels different containing agar LB on

Ϫ 0.25 oarabinose no ,

amgRS 0.5 eeinmutant. deletion [ µ 0.004 0.031 0.063 0.063 1.0 0.5 MIC g/mL] 5of15 Table S1. Tobramycin hypersensitive mutants Trans- Genomic ORF Reading Efficiency of ORF* Gene Allele† poson‡ position* position§ Frame¶ platingʈ MIC** Function*

Reference strains PAO1 — — — — — — 9.70E-01 1 PA3303 (Control) — M7 phoA 3701871 312 (392) (ϩ) 9.40E-01 1 Probable MFS transporter Regulation PA5199 amgS A3ϩ lacZ 5852306 84–85 ϩ1 1.1 E-05 0.125 Two-component (440) sensor G5 lacZ 5852204 118 (440) Ϫ3 7.4 E-06 0.063 J2 phoA 5852302 84–85 Ϫ1 2.1 E-06 0.125 (440) PA5200 amgR M7ϩ phoA 5853025 123 (248) ϩ3 2.4 E-05 0.063 Two-component response regulator F20 phoA 5853276 40 (248) Ϫ3 1.6 E-05 0.063 PA4398 — F11ϩ phoA 4929958 654–655 ϩ1 4.1 E-04 0.25 Two-component (699) sensor PA5471 — M5ϩ lacZ 6159941 252 (380) Ϫ2 5.4 E-06 0.25 Regulator PA0285 — F14 lacZ 319897 232 (761) ϩ3 1.9 E-02 0.5 EAL/GGDEF protein G5 lacZ 319164 477 (761) Ϫ3 1.8 E-02 0.5 PA0032 — B7 lacZ 34721 32 (305) Ϫ2 1.2 E-02 0.5 Regulator Proteases/ protein folding PA4942 hflK I15ϩ phoA 5546850 46–47 ϩ1 4.8 E-05 0.25 Protease subunit (401) PA5053 hslV L23 phoA 5692974 132–133 Ϫ1 5.6 E-05 0.5 Heat-shock (178) protease N18 phoA 5693044 156 (178) ϩ3 3.7 E-04 0.5 PA3649 mucP I8 phoA 4087704 391 (451) ϩ2 4.00E-04 0.5 Membrane protease PA3623 — B7 lacZ 4059640 90 (298) ϩ2 2.1 E-03 0.5 Membrane protease PA5254 fkbZ J20 lacZ 5916690 155–156 ϩ1 3.4 E-04 0.5 Peptidyl-prolyl (210) K10 lacZ 5916498 91–92 ϩ1 2.9 E-03 0.5 (210) Transport and efflux PA0427 oprM O1 lacZ 477135 267 (486) ϩ3 2.4 E-06 0.25 Efflux transporter C15 lacZ 477784 484 (486) Ϫ3 7.5 E-05 0.5 M5 lacZ 476892 186 (486) (ϩ) nd 0.25 PA2018 mexY B19ϩ lacZ 2209113 731 (1046) (Ϫ) 8.9 E-05 0.25 Efflux transporter H7 phoA 2210277 343 (1046) ϩ2 1.1 E-03 0.25 J24 lacZ 2209679 542 (1046) ϩ3 1.0 E-04 0.25 PA2019 mexX B16 lacZ 2212203 102–103 Ϫ1 2.7 E-07 0.25 Efflux (397) transporter PA5366 pstB H12 phoA 6033812 77 (278) ϩ2 2.3 E-04 0.25 Phosphate transport I11 phoA 6033582 154 (278) Ϫ3 1.3 E-04 0.25 PA5367 pstA E2 phoA 6035587 49 (559) ϩ3 6.3 E-05 0.5 Phosphate transport G5 phoA 6034685 349–350 Ϫ1 4.7 E-05 0.5 (559) PA5368 pstC C14 phoA 6036395 464 (678) ϩ3 3.5 E-04 0.5 Phosphate transport H17 phoA 6035880 636 (678) ϩ2 9.3 E-04 1 M9 phoA 6036925 288–289 ϩ1 1.0 E-03 0.5 (678) PA0016 trkA C3ϩ lacZ 18364 125 (458) ϩ2 7.0 E-05 0.25 Potassium transport

Lee et al. www.pnas.org/cgi/content/short/0903619106 6of15 Trans- Genomic ORF Reading Efficiency of ORF* Gene Allele† poson‡ position* position§ Frame¶ platingʈ MIC** Function*

I21 phoA 18646 30–31 Ϫ1 2.3 E-04 0.5 (458) J21 phoA 18097 213–214 Ϫ1 1.1 E-04 0.5 (458) PA3210 trkH E14 phoA 3599853 306–307 Ϫ1 1.5 E-03 0.5 Potassium (485) transport N7 phoA 3599149 72 (485) Ϫ2 9.0 E-02 0.5 PA5518 ybaL F6 lacZ 6210333 478 (568) ϩ2 2.7 E-04 0.5 Potassium efflux L7 lacZ 6210660 369 (568) ϩ2 2.1 E-03 0.5 PA0162 opdC A14 lacZ 185837 414 (445) ϩ3 5.1 E-04 0.5 Porin D20 lacZ 184625 10 (445) Ϫ2 2.00E-02 0.5 I6 lacZ 184759 55 (445) ϩ2 1.20E-03 0.5 PA0174 — N15 lacZ 198655 8–9 (201) Ϫ1 3.40E-03 0.5 Transporter PA0186 atsR H21 lacZ 212492 51–52 ϩ1 3.4 E-03 0.5 ABC transporter (354) N19 lacZ 213331 330 (354) ϩ3 4.0 E-03 0.5 PA0375 ftsX E16 lacZ 419595 11 (336) ϩ2 8.3 E-04 0.5 ABC Transporter I6 phoA 419895 111 (336) ϩ2 1.3 E-02 0.5 PA0426 mexB L6ϩ lacZ 473313 41 (1047) Ϫ3 3.3 E-04 0.25 Efflux transporter E9 lacZ 474122 310 (1047) ϩ3 4.3 E-04 0.5 G5 lacZ 474509 439 (1047) ϩ3 5.8 E-04 0.5 PA2494 mexF L18ϩ phoA 2811286 426 (1063) ϩ2 1.5 E-05 0.125 Efflux transporter E1 phoA 2811682 558 (1063) ϩ2 4.3 E-05 0.5 L20 lacZ 2812327 773 (1063) ϩ2 4.4 E-04 0.5 PA5250 ydgQ G18 phoA 5911566 151 (253) ϩ2 1.9 E-04 0.5 Transporter PA5479 gltP M21 lacZ 6170411 365–366 Ϫ1 9.2 E-04 0.5 Proton- (445) glutamate symporter K13 phoA 6170508 334 (445) Ϫ3 3.0 E-03 0.5 PA5504 — J21 lacZ 6196471 52 (226) ϩ2 2.9 E-03 0.5 ABC transporter PA0072 — N12 lacZ 85958 109–110 (ϩ) 1.1 E-02 0.5 ABC transporter (400) PA0073 — G6 lacZ 86767 79–80 ϩ1 1.2 E-03 0.5 (240) Metabolism PA0592 ksgA G13 lacZ 651207 57–58 Ϫ1 4.5 E-04 0.5 rRNA methylase (269) PA3686 adk L21ϩ lacZ 4126958 211–212 Ϫ1 5.2 E-04 0.25 Adenylate kinase (216) PA3013 foaB A23 lacZ 3373312 372 (392) ϩ2 2.9 E-05 0.25 Fatty acid oxidation K11 lacZ 3373605 274 (392) ϩ3 1.4 E-04 0.5 PA0195 pntA K18 lacZ 224776 225 (501) ϩ2 7.5 E-03 0.5 NAD/NADP transhydrogenase PA0265 gabD H20 lacZ 300565 348 (484) Ϫ3 1.2 E-03 0.5 Dehydrogenase PA0293 aguB C8 lacZ 330009 287 (293) Ϫ2 4.0 E-03 0.5 Polyamine metabolism PA0506 — F13 phoA 565313 132–133 Ϫ1 8.3 E-05 0.5 Acyl-CoA (602) dehydrogenase G1 phoA 565036 41–42 (ϩ) 1.0 E-04 0.5 (602) I13 phoA 566515 534 (602) Ϫ3 1.7 E-05 0.5 PA3175 — G9 lacZ 3565665 158–159 Ϫ1 2.3 E-01 0.5 (312) PA3823 tgt M4 phoA 4279947 186 (373) Ϫ2 1.1 E-03 0.5 tRNA modification O1 phoA 4280005 167 (373) ϩ2 3.8 E-01 0.5 O13 phoA 4280129 126 (373) Ϫ2, Ϫ3 5.3 E-04 1 PA4422 — A24 lacZ 4957372 70–71 ϩ1 7.5 E-04 0.5 Methylase (283)

Lee et al. www.pnas.org/cgi/content/short/0903619106 7of15 Trans- Genomic ORF Reading Efficiency of ORF* Gene Allele† poson‡ position* position§ Frame¶ platingʈ MIC** Function*

PA4693 pssA F16 lacZ 5271528 256–257 Ϫ1 9.2 E-05 0.5 Phosphatidyl (272) serine synthetase PA4979 — N13 lacZ 5592513 33 (387) Ϫ3 6.3 E-04 0.5 Acyl-CoA dehydrogenase PA5118 thiI G3 lacZ 5765726 139–140 Ϫ1 3.3 E-03 0.5 Thiamine (485) biosynthesis O4 lacZ 5765940 69 (485) Ϫ3 1.4 E-04 0.5 PA5245 yhbL K20 lacZ 5906972 116–117 ϩ1 1.3 E-02 0.5 Isoprenoid (223) biosynthesis PA5298 xpt B19 lacZ 5967194 162–163 Ϫ1 1.2 E-02 0.5 Purine synthesis (191) PA5349 — D20 lacZ 6017920 286 (385) Ϫ3 1.7 E-02 0.5 Rubredoxin reductase PA5417 soxD J8 lacZ 6096868 54 (107) ϩ2 3.0 E-03 0.5 Sarcosine oxidase Hypothetical PA3016 — F22ϩ phoA 3377948 118 (145) Ϫ2 6.6 E-05 0.125 Hypothetical A8 phoA 3378081 74 (145) ϩ2 9.3 E-05 0.125 PA5528 — G8ϩ phoA 6220519 73 (285) ϩ2 5.2 E-06 0.125 Hypothetical B15 phoA 6220115 208 (285) Ϫ3 1.2 E-04 0.5 PA4961 — E8 phoA 5569884 264–265 Ϫ1 1.9 E-04 0.25 Hypothetical (513) G4 phoA 5570052 320–321 Ϫ1 5.8 E-05 0.5 (513) PA0101 — B19 lacZ 122746 165–166 Ϫ1 1.4 E-03 0.5 Hypothetical (416) C9 lacZ 123114 289 (416) Ϫ3 1.5 E-03 0.5 O4 lacZ 122484 79–80 ϩ1 8.3 E-05 0.5 (416) PA0197 — B10 lacZ 227997 205 (271) ϩ2 3.6 E-03 0.5 Hypothetical PA0385 — C8 lacZ 427260 82–83 ϩ1 2.1 E-02 0.5 Hypothetical (108) PA3198 ypuG A7 lacZ 3591102 46 (251) ϩ3 1.7 E-02 0.5 Hypothetical PA5343 — H15 lacZ 6011638 89–90 Ϫ1 4.9 E-02 0.5 Hypothetical (284) N7 lacZ 6011313 198 (284) Ϫ2 4.0 E-03 0.5 O18 lacZ 6011490 139 (284) Ϫ2 1.3 E-03 0.5 PA5371 yciA K16 lacZ 6047141 76 (135) ϩ2 9.1 E-04 0.5 Hypothetical PA5383 yeiH H9 lacZ 6061429 30–31 ϩ1 7.0 E-04 0.5 Hypothetical (356) L14 lacZ 6061636 99–100 ϩ1 6.4 E-04 0.5 (356) Other PA1527 — B21 lacZ 1663564 717 (1163) Ϫ2 3.1 E-04 0.5 Chromosome segregation PA2463 — G3 lacZ 2779943 223 (566) Ϫ2 2.3 E-03 0.5 Protein export PA5493 polA L20 lacZ 6185563 320 (914) ϩ3 1.2 E-03 0.5 DNA polymerase I Intergenic PA0093/94 Ϫ/Ϫ N17 lacZ 114604 (Ϫ9), (ϩ),(ϩ) 3.3 E-03 0.5 Hypothetical/ (ϩ7) hypothetical PA0138/39 Ϫ/ahpC F7 lacZ 158181 (ϩ147), (ϩ),(ϩ) 1.8 E-03 0.5 Transporter/alkyl (Ϫ18) hydroperoxide reductase PA0326/27 Ϫ/Ϫ O8 lacZ 367332 (Ϫ203), (Ϫ),(Ϫ) 1.3 E-02 0.5 ABC (Ϫ125) transporter/ hypothetical PA3587/88 metR/opdR I7 lacZ 4020664 (ϩ2), (ϩ),(Ϫ) 1.0 E-01 0.5 Regulator/porin (ϩ4) PA3826/27 Ϫ/yjgQ B7 lacZ 4284318 (ϩ35), (ϩ),(Ϫ) 6.7 E-02 0.5 Hypothetical/ (ϩ97) hypothetical

Lee et al. www.pnas.org/cgi/content/short/0903619106 8of15 Trans- Genomic ORF Reading Efficiency of ORF* Gene Allele† poson‡ position* position§ Frame¶ platingʈ MIC** Function*

PA4914/15 Ϫ/Ϫ J12 lacZ 5514712 (ϩ43), (Ϫ),(Ϫ) 4.6 E-03 0.5 Regulator/ (Ϫ14) chemotaxis receptor PA5123/24 Ϫ/ntrB D22 lacZ 5772295 (ϩ269), (ϩ),(ϩ) 2.6 E-03 0.5 Hypothetical/ (Ϫ2) two-component sensor PA5429/30 aspA/Ϫ O8 lacZ 6111007 (ϩ324), (ϩ),(Ϫ) 4.0 E-04 0.5 Aspartate (ϩ40) / hypothetical PA5471/72 Ϫ/Ϫ D24 lacZ 6160742 (Ϫ44), (Ϫ),(Ϫ) 2.7 E-04 0.5 Regulator/ (ϩ324) hypothetical PA5529/30 — I3 lacZ 6223090 (ϩ233), (Ϫ),(Ϫ) 2.5 E-03 0.5 Potassium (Ϫ83) efflux/MFS transporter

The efficiency of plating on tobramycin agar and tobramycin minimal inhibitory concentration for all mutants exhibiting increased tobramycin sensitivity are presented. All transposon insertion sites were confirmed by resequencing. Alleles for which genetic linkage analysis confirmed the associated sensitivity phenotype are indicated, as are those genes for which only in-frame transposon insertions (encoding translational gene fusions) were identified. All mutants exhibiting at least a 2-fold decrease in the tobramycin minimal inhibitory concentration are included in the list. The genotype-phenotype associations were confirmed for nearly half of the genes (29/61) by the identification of multiple independent alleles, and for twelve additional alleles by genetic linkage analysis. *http://v2.pseudomonas.com/index.jsp †Tobramycin hypersensitivity confirmed by genetic linkage test. ‡lacZ,ISlacZ/hah; phoA,ISphoA/hah §Values correspond to the codon at which the insertion is situated and the total number of codons in the open reading frame ¶Value reflects the relative orientation and translational reading frame of the transposon-borne lacZ or phoA gene and the target gene, with Љϩ2Љ corresponding to an in-frame insertion. If no number is given, i.e., ЉϩЉ or Љ—Љ, the sequence information was ambiguous at the transposon insertion junction (although the orientation of the transposon was defined). In such cases, the locations of the insertion provided in columns 5 and 6 are estimates. ʈThe efficiency of colony formation on LB supplemented with 0.5 ␮g/mL tobramycin relative to unsupplemented LB is provided. **The minimal concentration of tobramycin inhibiting growth on LB agar is provided. Assay results were similar on Mueller-Hinton agar except the MIC values for all strains were lower by a factor of 2.

Lee et al. www.pnas.org/cgi/content/short/0903619106 9of15 Table S2. Functional categories of genes affected in tobramycin hypersensitive mutants Tobramycin hypersensitive Whole genome mutants

Functional class Genes Fraction Genes Fraction

Adaptation 175 3.1% 0 0.0% Amino acid biosynthesis and metabolism 233 4.2% 5 8.2% Antibiotic resistance and susceptibility 37 0.7% 5 8.2% Biosynthesis of cofactors, prosthetic groups and carriers 159 2.9% 1 1.6% Carbon compound catabolism 168 3.0% 2 3.3% Cell division 29 0.5% 2 3.3% Cell wall, LPS 152 2.7% 0 0.0% Central intermediary metabolism 99 1.8% 1 1.6% Chaperones and heat shock proteins 54 1.0% 2 3.3% Chemotaxis 64 1.1% 0 0.0% DNA metabolism 88 1.6% 1 1.6% Energy metabolism 206 3.7% 1 1.6% Fatty acid and lipid metabolism 62 1.1% 2 3.3% Membrane proteins 675 12.1% 17 27.9% Motility and attachment 112 2.0% 0 0.0% Nucleotide synthesis and metabolism 84 1.5% 2 3.3% Protein secretion 99 1.8% 0 0.0% Putative 473 8.5% 3 4.9% Related to phage, transposon plasmid 65 1.2% 0 0.0% Secreted factors 88 1.6% 0 0.0% Transcription, RNA processing 55 1.0% 2 3.3% Transcriptional regulators 474 8.5% 2 3.3% Translation, post-translational modification, degradation 195 3.5% 3 4.9% Transport of small molecules 594 10.7% 18 29.5% Two-component regulatory systems 121 2.2% 3 4.9% Hypothetical 2350 42.2% 20 32.8% TOTAL 5572 100.0% 61 100.0%

The tobramycin hypersensitive mutant group (Table S1) is compared to the whole genome. The genomic categories correspond to Pseudocap function classes (http://v2.pseudomonas.com/search.jsp). ЉMembrane proteinsЉ and ЉTransport of small moleculesЉ categories are highly represented and are in boldface.

Lee et al. www.pnas.org/cgi/content/short/0903619106 10 of 15 Table S3. Effect of the ⌬amgRS mutation on gene expression AmgRSϩ/AmgRS– ϩTob/–Tob

PA number Gene ϩ Tob – Tob AmgRSϩ AmgRS– Description

AmgRS-dependent genes amgR 1123.2 379.5 2.1 Ϫ1.4 two-component response regulator PA5200 PA5199 amgS 30.0 12.5 2.4 1.0 two-component sensor PA2405 20.9 3.6 Ϫ1.8 Ϫ10.6 hypothetical protein PA5217 10.4 12.0 Ϫ1.1 1.0 probable binding protein component of ABC iron transporter PA2830 htpX 7.6 2.9 3.4 1.3 heat shock protein PA1331 7.4 2.6 3.9 1.3 conserved hypothetical protein PA2404 5.6 2.5 Ϫ1.6 Ϫ3.6 hypothetical protein PA2403 5.5 3.4 Ϫ1.5 Ϫ2.4 hypothetical protein PA2549 5.4 1.7 5.8 1.8 conserved hypothetical protein PA3712 4.9 3.2 2.6 1.7 hypothetical protein PA5528 4.7 3.1 3.4 2.2 hypothetical protein PA5564 gidB 4.2 Ϫ1.1 Ϫ1.2 Ϫ5.3 glucose inhibited division protein B PA2408 4.1 2.4 Ϫ1.4 Ϫ2.5 probable ATP-binding component of ABC transporter PA2685 4.0 1.6 1.2 Ϫ2.1 conserved hypothetical protein PA1105 fliJ 3.9 Ϫ1.0 Ϫ1.4 Ϫ5.9 flagellar protein PA4277.3 3.7 2.5 Ϫ1.2 Ϫ1.9 tRNA Tyrosine PA2407 3.7 2.8 Ϫ1.3 Ϫ1.7 probable adhesion protein PA3575 3.7 2.3 2.6 1.6 hypothetical protein PA1882 3.7 1.6 2.6 1.2 probable transporter PA3154 wzy 3.7 1.2 Ϫ1.0 Ϫ3.2 B-band O-antigen polymerase PA3670 3.6 1.5 Ϫ1.0 Ϫ2.5 hypothetical protein PA5509 3.6 1.4 Ϫ1.3 Ϫ3.5 hypothetical protein PA1812 mltD 3.6 1.2 Ϫ1.1 Ϫ3.3 membrane-bound lytic murein transglycosylase D precursor PA0593 pdxA 3.6 1.2 Ϫ1.7 Ϫ5.1 pyridoxal phosphate biosynthetic protein PA0866 aroP2 3.6 1.0 Ϫ1.4 Ϫ5.0 aromatic amino acid transport protein PA3139.1 3.6 3.0 1.0 Ϫ1.2 tRNA Asparagine PA2049 3.6 1.1 1.3 Ϫ2.4 hypothetical protein PA2109 3.5 1.1 Ϫ1.9 Ϫ6.3 hypothetical protein PA4517 3.5 Ϫ1.0 Ϫ1.1 Ϫ3.9 conserved hypothetical protein PA0916 3.4 1.2 1.0 Ϫ2.9 conserved hypothetical protein PA2522 czcC 3.3 1.2 2.0 Ϫ1.4 outer membrane protein precursor PA0594 surA 3.3 1.1 Ϫ1.5 Ϫ4.6 peptidyl-prolyl cis-trans isomerase PA4662 murI 3.3 1.6 Ϫ1.5 Ϫ3.0 glutamate racemase PA4277.2 3.2 2.0 Ϫ1.1 Ϫ1.8 tRNA Glycine PA4581-PA4582 intergenic 3.2 2.5 1.1 Ϫ1.1 PA4581-PA4582 intergenic region PA5009 waaP 3.2 1.2 1.2 Ϫ2.2 lipopolysaccharide core biosynthesis protein PA3149 wbpH 3.2 1.2 Ϫ1.8 Ϫ4.8 probable glycosyltransferase PA5529 3.2 4.0 1.4 1.8 probable sodium/proton antiporter PA3608 potB 3.1 1.3 Ϫ1.3 Ϫ3.1 polyamine transport protein PA5007 3.1 1.0 Ϫ1.0 Ϫ3.1 hypothetical protein PA1073 braD 3.0 1.0 Ϫ1.3 Ϫ3.8 branched-chain amino acid transport protein PA5252 3.0 1.2 1.1 Ϫ2.2 probable ATP-binding component of ABC transporter PA0915 3.0 1.4 Ϫ1.5 Ϫ3.2 conserved hypothetical protein PA4817 3.0 1.0 1.1 Ϫ2.6 hypothetical protein PA3153 wzx 3.0 1.5 Ϫ1.5 Ϫ2.8 O-antigen PA2373 2.9 1.0 1.4 Ϫ2.1 conserved hypothetical protein PA3787 2.8 1.1 2.7 1.1 conserved hypothetical protein PA0920 2.8 Ϫ1.0 1.7 Ϫ1.7 hypothetical protein PA0382 micA 2.8 1.1 Ϫ1.2 Ϫ3.1 DNA mismatch repair protein PA4750 folP 2.7 Ϫ1.0 1.8 Ϫ1.6 dihydropteroate synthase PA5008 2.7 Ϫ1.0 1.0 Ϫ2.7 hypothetical protein PA1688 2.7 1.2 1.1 Ϫ2.2 hypothetical protein

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PA number Gene ϩ Tob – Tob AmgRSϩ AmgRS– Description

PA2733 2.7 1.1 Ϫ1.4 Ϫ3.5 conserved hypothetical protein PA4284 recB 2.7 1.1 Ϫ1.5 Ϫ3.5 exodeoxyribonuclease V beta chain PA1796.4 2.7 2.4 Ϫ1.0 Ϫ1.2 tRNA Histidine PA5479 gltP 2.7 1.1 Ϫ1.4 Ϫ3.5 proton-glutamate symporter PA4959 fimX 2.7 1.0 Ϫ1.2 Ϫ3.1 hypothetical protein PA1760 2.7 1.2 Ϫ1.4 Ϫ3.2 probable transcriptional regulator PA4480 mreC 2.7 1.3 Ϫ1.1 Ϫ2.2 rod shape-determining protein PA4663 moeB 2.7 1.2 Ϫ1.2 Ϫ2.6 molybdopterin biosynthesis protein PA2613 2.7 1.1 Ϫ1.1 Ϫ2.5 conserved hypothetical protein PA4558 2.6 1.1 Ϫ1.4 Ϫ3.3 probable peptidyl-prolyl cis-trans isomerase, FkbP-type PA4416 murF 2.6 1.2 Ϫ1.8 Ϫ4.0 UDP-N-acetylmuramoylalanyl-D- glutamyl-2, 6-diaminopimelate–D- alanyl-D-alanyl PA2732 2.6 1.2 Ϫ1.4 Ϫ3.1 hypothetical protein PA2643 nuoH 2.6 Ϫ1.2 Ϫ1.7 Ϫ5.1 NADH dehydrogenase I chain H PA4417 murE 2.6 1.0 Ϫ1.4 Ϫ3.5 UDP-N-acetylmuramoylalanyl-D- glutamate-2, 6-diaminopimelate ligase PA0924 2.6 1.1 1.2 Ϫ1.9 hypothetical protein PA1071 braF 2.6 1.1 Ϫ1.9 Ϫ4.7 branched-chain amino acid transport protein PA2909 2.6 Ϫ1.0 1.3 Ϫ2.1 hypothetical protein PA3636 kdsA 2.6 Ϫ1.0 Ϫ1.2 Ϫ3.2 2-dehydro-3-deoxyphosphooctonate aldolase PA5006 2.6 1.0 Ϫ1.1 Ϫ2.8 hypothetical protein PA5041 pilP 2.6 1.1 Ϫ1.4 Ϫ3.3 type 4 fimbrial biogenesis protein PA0667 2.6 1.0 Ϫ1.2 Ϫ2.9 conserved hypothetical protein PA5450 wzt 2.5 1.1 Ϫ1.2 Ϫ2.9 ABC subunit of A-band LPS efflux transporter PA4672 2.5 1.3 1.0 Ϫ1.9 peptidyl-tRNA PA3643 lpxB 2.5 1.2 Ϫ1.2 Ϫ2.7 lipid A-disaccharide synthase PA2604 2.5 1.8 2.5 1.8 conserved hypothetical protein PA4628 lysP 2.5 1.2 Ϫ1.5 Ϫ3.2 lysine-specific permease PA5003 2.5 1.1 Ϫ1.2 Ϫ2.8 hypothetical protein PA0976 2.5 1.1 1.1 Ϫ2.1 conserved hypothetical protein PA4479 mreD 2.5 1.2 Ϫ1.1 Ϫ2.4 rod shape-determining protein PA5487 2.5 1.1 Ϫ1.3 Ϫ2.9 hypothetical protein PA4292 2.5 1.3 Ϫ1.1 Ϫ2.1 probable phosphate transporter PA2640 nuoE 2.5 Ϫ1.1 Ϫ1.4 Ϫ3.7 NADH dehydrogenase I chain E AmgRS-repressed genes PA3127 Ϫ2.5 1.2 1.3 3.7 hypothetical protein PA0279 Ϫ2.5 1.0 1.5 3.9 probable transcriptional regulator PA0061 Ϫ2.5 1.0 1.4 3.7 hypothetical protein PA1744 Ϫ2.5 Ϫ1.3 Ϫ1.1 1.8 hypothetical protein PA4365 Ϫ2.5 Ϫ1.2 2.1 4.4 probable transporter PA0660 Ϫ2.6 Ϫ1.1 1.3 3.1 hypothetical protein PA1484 Ϫ2.6 1.1 1.3 3.7 probable transcriptional regulator PA1507-PA1508 intergenic Ϫ2.6 Ϫ1.0 Ϫ1.1 2.4 PA1507-PA1508 intergenic region PA3782 Ϫ2.6 1.1 1.3 3.7 probable transcriptional regulator PA0201 Ϫ2.6 Ϫ1.1 1.2 2.8 hypothetical protein PA0803 Ϫ2.6 1.0 Ϫ1.8 1.5 hypothetical protein PA3662 Ϫ2.6 1.2 1.8 5.9 hypothetical protein PA2306 Ϫ2.6 Ϫ1.1 1.2 2.8 conserved hypothetical protein PA2331 Ϫ2.6 Ϫ1.2 1.2 2.8 hypothetical protein PA4287 Ϫ2.6 1.4 2.0 7.4 hypothetical protein PA2799 Ϫ2.7 Ϫ1.1 1.3 3.1 hypothetical protein PA2452 Ϫ2.7 Ϫ2.3 Ϫ1.7 Ϫ1.5 hypothetical protein PA0468 Ϫ2.7 Ϫ1.4 1.4 2.8 hypothetical protein PA1885 Ϫ2.7 1.2 1.5 4.8 conserved hypothetical protein PA5150 Ϫ2.7 1.3 1.2 4.3 probable short-chain dehydrogenase PA5294 Ϫ2.7 Ϫ1.1 7.4 19.3 hypothetical protein PA4833 Ϫ2.8 Ϫ1.3 1.7 3.7 conserved hypothetical protein

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PA number Gene ϩ Tob – Tob AmgRSϩ AmgRS– Description

PA4918 Ϫ2.8 Ϫ1.1 2.1 5.2 hypothetical protein PA2766 Ϫ2.8 Ϫ1.4 2.5 5.1 probable transcriptional regulator PA0877 Ϫ2.8 Ϫ1.1 1.5 3.8 probable transcriptional regulator PA1136 Ϫ2.8 Ϫ1.1 1.3 3.1 probable transcriptional regulator PA2937 Ϫ2.8 Ϫ1.2 1.9 4.4 hypothetical protein PA4364 Ϫ2.8 Ϫ1.1 2.8 7.3 hypothetical protein PA1122 Ϫ2.8 Ϫ1.1 1.2 3.0 probable peptide deformylase PA0661 Ϫ2.8 Ϫ1.2 1.4 3.1 conserved hypothetical protein PA0656 Ϫ2.9 Ϫ1.1 1.6 4.2 probable HIT family protein PA3056 Ϫ2.9 Ϫ1.2 1.8 4.1 hypothetical protein PA5374 betI Ϫ2.9 1.0 2.9 8.7 transcriptional regulator PA2272 pbpC Ϫ2.9 Ϫ2.2 Ϫ1.2 1.1 penicillin-binding protein 3A PA3882 Ϫ2.9 1.0 1.2 3.7 hypothetical protein PA2719 Ϫ2.9 Ϫ1.9 3.9 6.0 hypothetical protein PA5206 argE Ϫ2.9 Ϫ1.4 1.6 3.4 acetylornithine deacetylase PA3161 himD Ϫ3.0 1.0 2.7 8.1 integration host factor beta subunit PA0738 Ϫ3.0 1.0 1.9 5.7 conserved hypothetical protein PA3338 Ϫ3.0 1.0 1.2 3.7 hypothetical protein PA2759 Ϫ3.0 Ϫ1.4 2.3 4.7 hypothetical protein PA3895 Ϫ3.0 Ϫ1.6 1.0 1.9 probable transcriptional regulator PA0200 Ϫ3.0 Ϫ3.3 2.0 1.8 hypothetical protein PA1518 Ϫ3.1 Ϫ1.0 1.7 5.0 conserved hypothetical protein PA2145 Ϫ3.1 Ϫ1.6 2.1 4.1 hypothetical protein PA4205 mexG Ϫ3.1 Ϫ1.2 2.3 6.2 hypothetical protein PA2433 Ϫ3.2 Ϫ1.4 2.3 5.3 hypothetical protein PA4336 Ϫ3.2 Ϫ1.2 1.1 3.0 conserved hypothetical protein PA1653 Ϫ3.2 1.2 1.5 5.8 probable transcriptional regulator PA2883 Ϫ3.2 1.1 1.6 5.8 hypothetical protein PA1321 cyoE Ϫ3.2 Ϫ2.5 Ϫ2.4 Ϫ1.8 cytochrome o ubiquinol oxidase protein PA5546 Ϫ3.2 Ϫ1.1 1.2 3.6 conserved hypothetical protein PA1203 Ϫ3.3 Ϫ2.3 1.4 2.0 hypothetical protein PA4990 Ϫ3.3 Ϫ1.3 2.0 5.0 SMR multidrug efflux transporter PA3731 Ϫ3.3 Ϫ1.2 2.0 5.6 conserved hypothetical protein PA5373 betB Ϫ3.4 Ϫ1.1 1.7 5.3 betaine aldehyde dehydrogenase PA4575 Ϫ3.5 1.1 1.4 5.3 hypothetical protein PA3572 Ϫ3.5 Ϫ2.7 1.1 1.5 hypothetical protein PA1029 Ϫ3.5 Ϫ1.2 2.1 6.0 hypothetical protein PA2845 Ϫ3.6 1.2 1.2 4.8 hypothetical protein PA3927 Ϫ3.6 Ϫ1.1 1.4 4.6 probable transcriptional regulator PA4026 Ϫ3.6 Ϫ1.1 2.2 7.1 probable acetyltransferase PA4114 Ϫ3.6 Ϫ1.2 3.2 10.1 spermidine acetyltransferase PA5172 arcB Ϫ3.8 Ϫ1.1 1.2 4.2 ornithine carbamoyltransferase, catabolic PA2816 Ϫ3.9 1.0 1.5 5.9 hypothetical protein PA1282 Ϫ4.0 Ϫ1.5 7.3 18.9 probable MFS transporter PA1414 Ϫ4.0 Ϫ2.7 Ϫ1.1 1.3 hypothetical protein PA1318 cyoB Ϫ4.0 Ϫ4.0 Ϫ2.0 Ϫ2.0 cytochrome o ubiquinol oxidase subunit I PA0737 Ϫ4.0 Ϫ1.0 3.1 11.7 hypothetical protein PA5460 Ϫ4.1 1.1 1.7 7.8 hypothetical protein PA1283 Ϫ4.1 Ϫ1.2 3.5 12.2 probable transcriptional regulator PA2622 cspD Ϫ4.2 Ϫ1.4 1.2 3.6 cold-shock protein PA1320 cyoD Ϫ4.5 Ϫ5.2 Ϫ1.8 Ϫ2.1 cytochrome o ubiquinol oxidase subunit IV PA2380 Ϫ4.6 Ϫ1.6 Ϫ1.3 2.3 hypothetical protein PA5158 Ϫ4.6 Ϫ2.2 Ϫ1.8 1.2 probable outer membrane protein PA2880 Ϫ4.6 Ϫ1.2 1.5 5.7 hypothetical protein PA1319 cyoC Ϫ4.6 Ϫ4.6 Ϫ2.2 Ϫ2.2 cytochrome o ubiquinol oxidase subunit III PA1747 Ϫ4.9 Ϫ1.0 1.6 7.8 hypothetical protein PA0109 Ϫ5.1 Ϫ4.0 4.0 5.1 hypothetical protein PA2804 Ϫ6.4 1.2 Ϫ1.3 5.9 hypothetical protein PA0160 Ϫ6.7 Ϫ1.1 1.4 9.0 hypothetical protein

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PA number Gene ϩ Tob – Tob AmgRSϩ AmgRS– Description

PA1317 cyoA Ϫ7.2 Ϫ4.2 Ϫ2.1 Ϫ1.2 cytochrome o ubiquinol oxidase subunit II PA4892 ureF Ϫ7.4 Ϫ4.2 1.1 1.9 accessory protein PA3619 Ϫ7.5 Ϫ1.1 Ϫ3.5 2.0 hypothetical protein PA1372-PA1373 intergenic Ϫ9.0 Ϫ4.7 Ϫ1.3 1.5 PA1372-PA1373 intergenic region PA4893 ureG Ϫ9.3 Ϫ8.1 1.0 1.2 urease accessory protein PA4894 Ϫ9.4 Ϫ6.7 Ϫ1.1 1.3 hypothetical protein PA5157 Ϫ12.6 Ϫ1.9 Ϫ1.5 4.4 probable transcriptional regulator PA4891 ureE Ϫ15.2 Ϫ8.8 1.9 3.2 urease accessory protein AmgRS-independent genes whose E. coli homologues are CpxRA-regulated PA0766 mucD (degP) –1.1 –1.1 –1.0 –1.1 periplasmic serine endoprotease PA5489 dsbA –1.4 1.0 1.3 1.9 periplasmic disulfide PA0486 (rdoA) –1.5 –1.2 1.5 1.9 Ser/Thr protein kinase PA2854 (ycfS) –1.3 –1.0 1.3 1.7 periplasmic protein of unknown function

All genes differing significantly (Ն 2.5-fold) in microarray hybridization between wild-type and ⌬amgRS strains in tobramycin-treated cultures (column 3) are included. Note that some genes (e.g., PA2405 and PA5217) exhibit AmgRS-dependent expression but are not induced by tobramycin exposure. Several genes whose E. coli homologues show strong cpxAR-dependent expression but appear to be relatively unaffected by loss of amgRS are also included at the end of the list (with the homologue gene names listed parenthetically). Tob, tobramycin

Lee et al. www.pnas.org/cgi/content/short/0903619106 14 of 15 Table S4. Aberrant cytoplasmic membrane proteins enhance tobramycin sensitivity and are toxic to ⌬amgRS mutants

Insertion Tobramycin MIC§ Nalidixic acid MIC§ Relative eop¶ position mexF allele (frame)* Transposon† Fusion site‡ No plasmid ϩmexF plasmid No plasmid ϩmexF plasmid amgRSϩ ⌬amgRS

Wild-type — — – 0.5 0.5 512 512 8.0E-01 8.7E-01 N21 47 (OF) lacZ – 1.0 0.5 64 512 1.2E ϩ 00 1.1E ϩ 00 F2 196 (OF) lacZ – 1.0 1.0 64 512 8.2E-01 1.6E ϩ 00 I17 247 (IF) phoA P1 1.0 0.5 64 512 8.0E-01 7.7E-01 H14 276 (IF) phoA P1 1.0 0.5 64 512 1.2E ϩ 00 1.1E ϩ 00 E18 343 (OF) phoA – 1.0 0.5 64 512 7.7E-01 8.7E-01 G8 414 (IF) phoA TM4 0.125 0.063 64 512 6.9E-01 2.0E-05 G15 426 (IF) phoA C3 0.125 0.063 64 512 8.5E-01 1.6E-05 L18 426 (IF) phoA C3 0.125 0.063 64 512 6.1E-01 1.7E-05 O24 506 (IF) phoA C4 0.25 0.125 64 512 8.5E-01 4.2E-04 E1 558 (IF) phoA TM7 0.25 0.25 64 512 6.2E-01 7.0E-05 N17 594 (IF) phoA P4 0.5 0.5 64 512 7.5E-01 6.6E-01 H11 621 (IF) phoA P4 0.5 0.5 64 512 7.6E-01 9.8E-01 M7 684 (IF) phoA P4 0.5 0.5 64 512 7.3E-01 9.7E-01 C4 938 (IF) phoA TM10 0.25 0.25 64 512 8.3E-01 6.3E-05

Strains carrying mexF insertion mutations in wild-type and ⌬amgRS genetic backgrounds were assayed for tobramycin sensitivity and plating efficiency. The in-frame insertions encode mexF-phoA hybrid proteins targeted to the cytoplasmic membrane, where they presumably fail to fold normally due to missing transmembrane sequences. Six of the insertions encoding such hybrid proteins (boldface type) enhanced tobramycin sensitivity in both wild-type and ⌬amgRS backgrounds and failed to form colonies efficiently in the ⌬amgRS genetic background at neutral pH. All of the double mutants were found to grow on acidic (pH 6.5) medium, although the toxic hybrid proteins increased tobramycin sensitivity under these conditions. *The transposon insertion site (of 1063 codons total) and whether the insertion generates an in-frame gene fusion are indicated. All fusions were resequenced to confirm transposon locations. OF, out-of-frame; IF, in-frame †lacZ,ISlacZ/hah; phoA,ISphoA/hah ‡The predicted subcellular localization of the in-frame fusion junction sites based on the predicted 12-span MexF topology are provided (Phobius algorithm; http://www.ebi.ac.uk/Tools/phobius/). P1, periplasmic domain 1; TM4, transmembrane domain 4; C3, cytoplasmic domain 3; etc. §Measured on LB (pH 6.5) ¶Efficiency of plating on LB pH 7.6 medium relative to LB pH 6.5 medium (pH 6.5 was permissive for growth and colony formation for all of the strains); eop, efficiency of plating

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