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 substrate-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 4.0 1.0 0.25 MIC (µg/ml) wt wt/+ /+ R R/+ S S/+ Genotype Fig. S1. Complementation of amgRS mutants. The tobramycin sensitivities of wild-type (MPAO1), ⌬amgRS, and amgR and amgS transposon insertion mutants are presented in strains lacking or containing plasmid p(amgRSϩ). The presence of the plasmid in the different strains elevated tobramycin resistance 2- to 4-fold over that observed for plasmid-free MPAO1, suggesting that an increased level of the regulator increases intrinsic resistance to the antibiotic. wt, MPAO1; ϩ,p(amgRSϩ); ⌬, ⌬amgRS;R,amgR::ISphoA/hah M7; S, amgS::ISlacZ/hah A3 Lee et al. www.pnas.org/cgi/content/short/0903619106 2of15 Tobramycin [µg/mL] MIC 0 2 4 8 16 32 64 128 256 512 2048 1024 [µg/mL] MPAO1 / pRmtD { 512 Control IS / pRmtD { 512 amgRS / pRmtD { 64 amgR::IS / pRmtD { 64 Fig. S2. Enhanced tobramycin sensitivity of amgRS mutant derivatives of a strain expressing a 16S rRNA methylase resistance function. The image shows duplicate tobramycin MIC determinations of strains carrying the rmtD gene (in plasmid pRmtD). The ⌬amgRS mutation reduced the MIC of strains expressing the resistance function 8-fold. MPAO1, parent; Control IS, strain carrying a PA3303 ISphoA/hah insertion which does not change aminoglycoside sensitivity; amgR::IS, amgR::ISphoA/hah M7. Lee et al. www.pnas.org/cgi/content/short/0903619106 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 Tobramycin [µg/mL] Arabinose MIC µ 0.5 0.004 0.008 0.016 0.031 0.063 0.125 0.25 pIbsC Induction 0 [ g/mL] + 0.5 MPAO1 + 1.0 + + 0.063 + 0.063 amgRS + 0.031 + + 0.004 Fig. S4. Enhanced tobramycin sensitivity of P. aeruginosa expressing the ibsC gene from E. coli. Growth of P. aeruginosa on LB agar containing different levels of tobramycin are shown. Induction of ibsC by arabinose addition resulted in an 8-fold reduction in tobramycin MIC for MPAO1 and the amgRS deletion mutant. Growth after 48 h is shown. pIbsC: ϩ, plasmid carrying Pbad ibsC; Ϫ, empty vector. Arabinose induction: ϩ, 0.025% arabinose; Ϫ, no arabinose Lee et al. www.pnas.org/cgi/content/short/0903619106 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) isomerase 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.