JOURNAL OF BACTERIOLOGY, Sept. 2011, p. 4790–4797 Vol. 193, No. 180021-9193/11/$12.00 doi:10.1128/JB.05133-11Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Membrane Proteases and Aminoglycoside Antibiotic Resistance�†Aaron Hinz,‡ Samuel Lee, Kyle Jacoby, and Colin Manoil*

Department of Genome Sciences, University of Washington, Seattle, Washington 98195

Received 26 April 2011/Accepted 1 July 2011

We present genetic studies that help define the functional network underlying intrinsic aminoglycosideresistance in Pseudomonas aeruginosa. Our analysis shows that proteolysis, particularly that controlled by themembrane protease FtsH, is a major determinant of resistance. First, we examined the consequences ofinactivating genes controlled by AmgRS, a two-component regulator required for intrinsic tobramycin resis-tance. Three of the gene products account for resistance: a modulator of FtsH protease (YccA), a membraneprotease (HtpX), and a membrane protein of unknown function (PA5528). Second, we screened mutationsinactivating 66 predicted proteases and related functions. Insertions inactivating two FtsH protease accessoryfactors (HflK and HflC) and a cytoplasmic protease (HslUV) increased tobramycin sensitivity. Finally, wegenerated an ftsH deletion mutation. The mutation dramatically increased aminoglycoside sensitivity. Many ofthe functions whose inactivation increased sensitivity appeared to act independently, since multiple mutationsled to additive or synergistic effects. Up to 500-fold increases in tobramycin sensitivity were observed. Most ofthe mutations also were highly pleiotropic, increasing sensitivity to a membrane protein hybrid, several classesof antibiotics, alkaline pH, NaCl, and other compounds. We propose that the network of proteases providesrobust protection from aminoglycosides and other substances through the elimination of membrane-disruptivemistranslation products.

The intrinsic antibiotic resistance of Pseudomonas aerugi-nosa and several other Gram-negative pathogens limits theeffective treatment of infection (16, 27). Although efflux pumpsand modifying functions contribute to the resistance (23, 26), ithas become apparent that more general stress response func-tions can be of equal or greater importance (9, 14, 24).

Two aminoglycosides, tobramycin and gentamicin, areamong the most valuable antibiotics for treating chronic P.aeruginosa infections (13). Aminoglycosides interfere withtranslation by increasing the frequency of misreading and byblocking the recycling of ribosomal subunits (4). Tai and Davis(6, 7) proposed that the translational errors lead to misfoldedproteins that compromise the cytoplasmic membrane barrier.The reduced barrier function enhances antibiotic uptake, lead-ing to the inhibition of translation and bacterial death.

We previously identified a two-component regulator of P.aeruginosa (AmgRS) whose inactivation led to a striking hy-persensitivity to tobramycin (24). Further analysis of AmgRSindicated that it regulates an envelope stress response akin tothe Escherichia coli CpxRA response (8, 28, 29). In the workreported here, we identify the AmgRS-regulated genes (andother genes) responsible for intrinsic aminoglycoside resis-tance. Our findings imply that proteolysis plays a crucial roleand suggest that the AmgRS response protects cells from ami-noglycosides by eliminating membrane-disruptive polypeptidesresulting from translational misreading.

MATERIALS AND METHODS

Strains, plasmids, and media. The strains used in this study are listed in TableS1 in the supplemental material. The reference strain of P. aeruginosa (MPAO1),the unmarked �amgRS mutation, and the defined transposon mutant library ofMPAO1 have been described (19, 24). The transposons used to create themutant library (ISphoA/hah and ISlacZ/hah) confer tetracycline resistance (19).Complementation plasmids were constructed by cloning promoter and openreading frame (ORF) sequences amplified from MPAO1 into a derivative ofpUCP19, a medium (�10 to 25) copy number plasmid (31). The followingsequences were represented (genome coordinates): htpX (3182591 to 3183982),yccA (2947420 to 2948608), and PA5528 (6220995 to 6219609). The minitrans-poson 7 (mTn7) derivative, carrying ftsH under araBAD promoter control, wasconstructed by cloning an araC-PBAD-ftsH fragment (carrying sequence from 119bp upstream to 26 bp downstream of the ftsH coding sequence) into a derivativeof mTn7T carrying a gentamicin resistance determinant (5 and data not shown).The �ftsH mutation (deleted of coding sequence from the 16th codon from the5� end to the 3rd codon from the 3� end) was generated using a standard method(17) with the growth of bacteria on LB-morpholineethanesulfonic acid (MES)(pH 6.0).

LB contained (per liter) 10 g Bacto-tryptone, 5 g yeast extract, 8 g NaCl,and 15 g agar. Buffered LB contained (per liter) 0.1 M buffer (morpho-linepropanesulfonic acid [MOPS], MES, 4-(2-hydroxyethyl)-1-piperazinepro-panesulfonic acid [EPPS], [N-tris(hydroxymethyl)methyl]-3-aminopropanesulfo-nic acid [TAPS], or 3-cyclohexylamino)-2-hydroxy-1-propanesulfonic acid[CAPSO]), 10 g Bacto-tryptone, 5 g yeast extract, and 15 g agar. The pH ofLB-MOPS, LB-EPPS, and LB-TAPS was adjusted with NaOH, and NaCl wasadded to bring the total sodium ion concentration to that of LB (0.137 M) or toa different level specified in the text. LB-MES was adjusted to pH 6.0 with KOHwith no added salt.

Construction of multiple mutants. Mutants carrying multiple transposon in-sertions were generated using a technique analogous to that described previouslyfor Francisella novicida (10). Transposon insertions (ISphoA/hah and ISlacZ/hah) were converted into 189-bp insertions by Cre recombination at loxP se-quences at the ends of the transposons (2). This recombination eliminates thetetracycline resistance markers carried on the transposons and makes it possibleto introduce additional mutations by recombination following transformationwith chromosomal DNA. The process was used to generate double, triple, andquadruple mutants.

For Cre recombination at loxP sites, a plasmid encoding Cre recombinase(pCre1) was introduced into PAO1 transposon mutants by conjugation with E.coli SM10�pir (2). Exconjugates were screened for the loss of the ISphoA/hahand ISlacZ/hah tetracycline resistance genes. That the appropriate recombina-

* Corresponding author. Mailing address: Department of GenomeSciences, University of Washington, Campus Box 355065, 1705 NEPacific St., Seattle, WA 98195. Phone: (206) 543-7800. Fax: (206)685-7301. E-mail: manoil@u.washington.edu.

† Supplemental material for this article may be found at http://jb.asm.org/.

‡ Present address: Department of Biochemistry, Microbiology andImmunology, University of Ottawa, Ottawa, Ontario, Canada.

� Published ahead of print on 15 July 2011.

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tion events had occurred in tetracycline-sensitive derivatives was confirmed bythe PCR amplification of the regions flanking the insertion sites. Additionaltransposon insertion mutations were introduced into the recombinants usingphage lambda Red recombination (11). Chromosomal DNA from a transposoninsertion mutant was electroporated into strains carrying plasmid pNB1, whichencodes lambda Red functions. Recombinants were selected on LB supple-mented with 60 �g/ml tetracycline, and the insertion positions were verified bysequencing the transposon junctions. Mutants normally were cured of the pNB1plasmid following construction. In mutants carrying three or more loxP sitessubjected to Cre recombination, there was a risk of recombination between loxPsites associated with different genes. Such recombination should lead to chro-mosomal inversions or deletions, depending on the orientation of the loxP sites.To avoid such rearrangements, the multiple mutants were constructed usingtransposon insertions with loxP sites in the same chromosomal orientation.Recombination between distant sites should lead to large lethal deletions, andthe corresponding strains usually should be inviable. Of seven multiple mutantsconstructed following this strategy that were checked by PCR, no inappropriaterearrangements were detected.

Antibiotic sensitivity assays. Strains were grown overnight at 37°C in 96-wellformat in LB lacking NaCl, diluted 100-fold in the same medium, and incubatedfor 90 min at 37°C. These conditions resulted in relatively uniform cell densitiesfor the strains, some of which grew slower than the wild type in LB containingNaCl. Aliquots (8 �l; �105 cells) were spotted on LB-MOPS (pH 7.6) agarcontaining antibiotics. The plates then were incubated for 24 h at 37°C andexamined for bacterial growth. The MIC was defined as the lowest antibioticconcentration preventing the visible lawn growth of the spot of cells.

Phenotype microarray assays. Phenotypic microarray analysis followed estab-lished protocols (35). Colonies were scraped from agar plates consisting ofsalt-free LB (1:5) and suspended in inoculating fluid (IF-0) to 85% transmit-tance. Cell suspensions were further diluted 1:200 in liquid medium containingsalt-free LB (1:5), and tetrazolium violet was added (1:100). Four phenotypicmicroarray plates (PM9, PM10, PM11C, and PM12B) were inoculated and in-cubated for 48 h at 37°C in an OmniLog plate reader to quantify the rate oftetrazolium reduction.

Screen of protease mutants for enhanced tobramycin sensitivity. A set ofmutants corresponding to 66 of 71 predicted proteases and protease accessoryfactors (based on clusters-of-orthologous-groups assignments at http://v2.pseudomonas.com/searchBoolean.jsp) was assembled from the MPAO1 trans-poson mutant library and arrayed in 96-well format (19). This set includes allproteases for which mutants were available in the mutant library, and for most ofthe genes two different mutants were included. To identify tobramycin-sensitivemutants, overnight cultures of the array of strains grown in LB were spotted ontotest plates containing subinhibitory tobramycin (0.125 �g/ml) or control plateslacking antibiotic. Dilutions of the cultures were spotted, making it possible todetect modest increases in sensitivity as reduced colony sizes. Such small differ-ences might have been missed in previous screens based on scoring the confluentgrowth of spots of cells.

The genes (alleles) represented in the mutant array are the following: PA0015(D4, E2), PA0277 (E4, G11), pfpI (A7, G1), PA0451 (A10, C9), slp (E4, F9), PA0639(A6, F10), PA0666 (B7), mucD (A9, G11), PA0779 (A7, A8, C3, G11), PA1005 (C4,D1), yedU (D6), PA1242 (A12, G9), aprD (D11, E4), PA1304 (C9, E1), PA1732 (A8,D5), PA1733 (B4, E5), PA1791 (G10, H8), clpP (A5), lon (F7), sohB (A8, E7),PA2044 (F9, F10), PA2102 (B9), PA2189 (F6), PA2283 (F8, F11), PA2286 (H10),PA2437 (H8), PA2438 (B5, H5), PA2439 (A11, D11, E11), PA2529 (A12, D4),PA2530 (A4, D4), PA2719 (D7, H4), eco (B10, G11), PA2814 (C1), htpX (C13, D8,H2), PA2873 (D4, F10), PA2973 (E4, H6), prc (D1, D12), hasD (E5, F12), mucP(C2, D3, I8), lasB (F6, F10), yhbV (B3, D2), yhbU (A5, G3), PA4012 (H7, H9),PA4048 (C12, G1), PA4171 (C3, C8), PA4295 (A6, E11), PA4336 (F4), algW (B12,F5, J3), pmbA (A5, D3), tldD (G7, G10), PA4576 (E10, F2), PA4582 (D9, E8), radA(D2, H10), PA4632 (B4, H9), PA4717 (A9, F1), PA4926 (A3, G6), hflC (C7, G4),hflK (D1, H6, I2, I15), PA4965 (F7, G9), PA5047 (B3, C8, F10), hslV (A4, E12, G7),hslU (B4, D2), PA5132 (B4, D7, G5), ctpA (E7, F7, G12), ybbJ (C6), and yegQ(B6, G3).

RESULTS

AmgRS-regulated genes contributing to intrinsic tobramy-cin resistance. We sought to identify the basis of AmgRS-mediated intrinsic tobramycin resistance. Transcriptional pro-filing had identified nine genes whose expression was stronglydependent on AmgRS in tobramycin-treated cells (24), and we

assumed that a subset of these genes was responsible for re-sistance. Strains with transposon insertions interrupting sevenof the genes were individually examined for tobramycin sensi-tivity. The mutants were retrieved from the sequence-definedtransposon library of strain PAO1 (19), their transposon loca-tions verified by sequencing, and their tobramycin sensitivitiesassayed (Table 1 and Fig. 1). Mutations in only two of thegenes (PA5528 and yccA) increased tobramycin sensitivity, and(with the exception of one allele) the decreases in MIC wereonly 2-fold. The exceptional insertion (PA5528 G8) generatesan in-frame PA5528-phoA gene fusion that we suspect in-creases tobramycin sensitivity through a gain-of-function effectanalogous to that observed for other membrane-directed hy-brid proteins (i.e., some MexF-PhoA hybrids) (24).

Functional redundancy underlies AmgRS-mediated tobra-mycin resistance. The weak single-mutant phenotypes mightreflect redundant protection from tobramycin by AmgRS-reg-ulated functions. To test this possibility, we examined the phe-notypes of mutants lacking multiple functions. Double, triple,and quadruple insertion mutants were generated using an it-erative technique involving phage lambda red recombination(Materials and Methods) (10), and their tobramycin sensitivi-ties were determined (Table 2 and Fig. 1). We first introducedadditional mutations into an htpX mutant background, sincehtpX exhibited the greatest AmgRS-dependent expression ob-served in a previous study (24). Of six double mutants exam-ined, two (htpX-PA5528 and htpX-yccA) exhibited greater sen-sitivities than expected from the additive effects of the parentalsingle mutants (i.e., MICs for mutants were reduced 4-foldrather than 2-fold relative to the MIC for the wild type). Asynergistic increase in sensitivity relative to that of the parentsalso was observed for the PA5528-yccA double mutant (Table2 and Fig. 1). The synergistic effects suggest that htpX, PA5528,and yccA contribute to tobramycin intrinsic resistance in apartially redundant fashion.

As a further test, we constructed a triple mutant carryinginsertions inactivating htpX, PA5528, and yccA. The triple mu-tant was extremely sensitive to tobramycin; its tobramycin MICwas 32-fold lower than that for the wild type and 2-fold lowerthan that for the amgRS deletion mutant (Table 2 and Fig. 1).The finding that the triple mutant was more sensitive than theamgRS mutant suggests that residual protection from tobra-mycin is provided by the basal expression of one or more of thethree genes in the amgRS-minus strain. Two other AmgRS-regulated genes (yegH and yebE) also may contribute to intrin-sic tobramycin resistance, since inactivating these genes in-creased the tobramycin sensitivity of the htpX-PA5528 andhtpX-yccA double mutants (Table 2 and data not shown). How-ever, the effects were small compared to those of combiningthe htpX, PA5528, and yccA mutations and were not observedin the htpX-PA5528-yccA mutant background (Table 2). Fur-thermore, the introduction of several additional mutations inAmgRS-regulated genes or amgR itself into the htpX-PA5528-yccA triple mutant did not increase tobramycin sensitivity fur-ther (Table 2 and data not shown). Thus, it appears that htpX,PA5528, and yccA are the primary AmgRS-regulated genesresponsible for intrinsic tobramycin resistance.

In complementation tests, plasmids carrying wild-type ver-sions of htpX, PA5528, or yccA each increased the tobramycinresistance of the htpX-PA5528-yccA triple mutant (Fig. 2). The

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rescue by the PA5528 plasmid was less complete than ex-pected. This result is presumably a consequence of PA5528overexpression, since the PA5528 plasmid increased the to-bramycin sensitivity of the control strain (designated theinsertion sequence [IS] control). The three plasmids alsoincreased the tobramycin resistance of the amgRS mutant(Fig. 2), further implying that the reduced expression ofthese genes in amgRS mutants underlies their enhancedtobramycin sensitivity.

The three genes we identified nearly completely account forthe role of AmgRS in tobramycin intrinsic resistance. Althoughnone of the genes have been characterized in P. aeruginosa, theE. coli homologues of htpX and yccA encode proteins associ-ated with membrane proteolysis. HtpX is a membrane-boundprotease (30, 32), whereas YccA modulates the activity of asecond membrane-bound protease, FtsH (18, 21, 34). The re-sults thus imply that proteolysis plays a critical role in tobra-mycin intrinsic resistance.

TABLE 1. Tobramycin resistance of mutants lacking AmgRS-regulated functions

MutationaAmgRS

dependenceb

(fold)Functiona Allelec Transposond Position in ORFe Reading

framefTobramycin MIC

(�g/ml)g

None (MPAO1) 0.5PA3303 (control) M7 phoA 312 (392) (�) 0.5amgR (PA5200) Two-component regulator M7� phoA 123 (248) �3 0.031�amgRS Two-component regulator/sensor Deletion 0.031htpX (PA2830) 7.6 Membrane protease C13� lacZ 36 (292) �1 0.5

K12� lacZ 68 (292) �2 0.5yegH (PA1331) 7.4 Putative transporter C5� lacZ 14 (516) �1 0.5

H5 lacZ 203 (516) �1 0.5yebE (PA3712) 4.9 Hypothetical protein L18� lacZ 120 (232) �3 0.5PA5528 4.7 Hypothetical protein D5� phoA 14 (285) �2 0.25

G8� phoA 73 (285) �2 0.063B15� phoA 208 (285) �3 0.25B24� lacZ 220 (285) �1 0.25

yceJ (PA3575) 3.7 Putative cytochrome P10� lacZ 46 (177) �2 0.5nlpD (PA3787) 2.8 Membrane protease H8� phoA 6 (283) �1 0.5yccA (PA2604) 2.5 Modulator of FtsH protease E3� lacZ 50 (223) �3 0.25

A13� phoA 134 (223) �1 0.5h

a See http://v2.pseudomonas.com/index.jsp. The so-called control strain carries an insertion (PA3303) that does not alter tobramycin sensitivity. The AmgRS-dependent genes were identified previously (24). Mutations in two AmgRS-dependent genes (PA1882 and PA2549) were not included, because they were notrepresented or were not successfully recovered from the PAO1 transposon library.

b Expression ratios of the wild type relative to �amgRS in tobramycin-treated cultures determined by microarray analysis (24).c A plus sign indicates that the mutation was recombined (by transformation) into MPAO1 to confirm linkage between the transposon insertion and tobramycin

sensitivity.d Transposons ISlacZ/hah and ISphoA/hah encode tetracycline resistance and carry lacZ and phoA sequences at their ends, respectively (19).e Values indicate the codon where the insertion is located versus the total number of codons (in parentheses) in the open reading frame (ORF).f Corresponds to the orientation and translational reading frame of the transposon-borne lacZ or phoA gene relative to the target gene, with �2 defining an in-frame

insertion.g The MIC of tobramycin preventing growth on LB-MOPS (pH 7.6) agar after 24 h (n � 3).h The yccA-A13 mutation increased tobramycin sensitivity when combined with htpX and/or PA5528 mutations (not shown), and its lack of an effect in a wild-type

genetic background may be due to the incomplete inactivation of YccA.

FIG. 1. Mutations in three AmgRS-regulated genes increase tobramycin sensitivity. Overnight cultures of the indicated strains were diluted100-fold and spotted on LB-MOPS (pH 7.6) agar containing tobramycin. Images were recorded after 24 h at 37°C. The increased sensitivity of thetriple mutant relative to the amgR mutant presumably reflects the basal transcription of htpX, yccA, and/or PA5528 in the mutant background. Thefollowing strains were tested (see Table S1 in the supplemental material): AHP36, AHP83, AHP124, AHP125, AHP147, AHP146, AHP145,AHP127, and AHP126.

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Contribution of additional protease factors to intrinsictobramycin resistance. To screen for other proteases contribut-ing to tobramycin resistance, we assembled a panel of P. aerugi-nosa mutants corresponding to 66 of 71 genes predicted toencode proteases and related functions (Materials and Meth-ods). For each gene, an average of two strains was retrievedfrom the MPAO1 transposon library (19). Mutants with in-creased sensitivity to tobramycin were identified by their re-

duced colony size on medium containing subinhibitory concen-trations of tobramycin. This assay should be more sensitivethan one based on the confluent growth of spots of cells (24).Insertions in four open reading frames increased sensitivity(hslU, hslV, hflK, and hflC) (Table 3). All of the genes had beenidentified previously (12, 24, 33). The mutations increased sen-sitivity modestly in both the wild-type and �amgRS geneticbackgrounds (Table 3). The hflK and hflC genes encode factorsregulating the specificity of FtsH membrane protease, and hslVand hslU encode a cytoplasmic heat shock protease (18, 25).The combined results of this and the previous screens suggestthat most of the nonessential protease activities that contributeto tobramycin intrinsic resistance have been identified.

Deletion of ftsH increases tobramycin sensitivity. Mutationsinactivating three factors (YccA, HflC, and HflK) that interactwith FtsH in E. coli increase tobramycin sensitivity. The role ofFtsH in P. aeruginosa had not been examined directly in theearlier screens, because insertions in the gene are absent fromthe mutant library, presumably because, as in E. coli (18), ftsHis essential for growth. To test whether FtsH contributes toaminoglycoside intrinsic resistance, we constructed a strainexpressing wild-type ftsH under arabinose promoter control(see Materials and Methods). In the course of these experi-ments, we discovered that although ftsH mutants grew slowlyon standard LB agar, FtsH was dispensable on LB lackingadded NaCl. This medium previously had been found to en-hance the growth of amgRS mutants (24). We thus constructedan ftsH deletion mutant (in the absence of a regulated wild-

TABLE 2. Tobramycin sensitivities of multiple mutantsa

MutantAllele for AmgRS-regulated gene Tobramycin MIC

(�g/ml)bhtpX yegH yebE PA5528 yceJ nlpD yccA

Wild type � � � � � � � 0.5

�amgRS � � � � � � � 0.031

Single mutants C13 � � � � � � 0.5� C5 � � � � � 0.5� � L18 � � � � 0.5� � � B15 � � � 0.25� � � � P10 � � 0.5� � � � � H8 � 0.5� � � � � � E3 0.25

Double mutants C13* C5 � � � � � 0.5C13* � L18 � � � � 0.5C13* � � B15 � � � 0.125C13* � � � P10 � � 0.5C13* � � � � � E3 0.125

� � � B15* � � E3 0.063

Triple mutants C13* C5 � B15* � � � 0.063C13* � L18 B15* � � � 0.063C13* � � B15* � � E3 0.016

Quadruple mutants C13* C5 � B15* � � E3* 0.016C13* � L18 B15* � � E3* 0.016C13* � � B15* P10 � E3* 0.016C13* � � B15* � H8 E3* 0.016

a The genotypes and tobramycin sensitivities of mutants with insertions in multiple AmgRS-regulated genes are shown. Mutant alleles are described in Table 1, andi63 insertions (generated from the corresponding transposon insertion by Cre recombination) are indicated by an asterisk. Multiple direct comparisons showed thattobramycin MICs for i63 mutants were equivalent to those for the corresponding transposon insertion mutants (not shown). �, Wild-type allele.

b MICs were determined on LB-MOPS (pH 7.6) agar and represent the consensus of at least three independent assays for each strain.

FIG. 2. Complementation of tobramycin-sensitive strains. Thetobramycin sensitivities of strains carrying plasmid clones of the indi-cated genes were assessed on LB-MOPS (pH 7.6) agar containingcarbenicillin (150 �g/ml). MICs were confirmed in duplicate assays.The IS control carries a transposon insertion in PA3303 which does notaffect tobramycin resistance.

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type gene) under permissive conditions to analyze the role ofthe protease in tobramycin intrinsic resistance.

The ftsH deletion mutation led to a dramatic increase in tobra-mycin sensitivity, about the same as that observed for amgRSmutations at pH 7.6 (Table 4). Unlike amgRS mutations, theeffect of the ftsH mutation was not greatly attenuated at pH 7.0(Table 4). The ftsH deletion was complemented by the expressionof the wild-type gene. The result shows that FtsH activity is acritical determinant of tobramycin intrinsic resistance.

To test whether FtsH and AmgRS contribute independentlyto tobramycin resistance, we constructed a double mutant lack-ing both activities (�ftsH amgR::i63). The strain was extremelysensitive to elevated pH but could be grown and analyzed atpH 7.0. Under these conditions, the strain was exquisitely sen-sitive to tobramycin, exhibiting a 500-fold decrease in MICrelative to that of the parent (Table 4). The results indicatethat FtsH and AmgRS make independent contributions to

intrinsic tobramycin resistance. PA5528 makes the greatestcontribution of the three AmgRS-regulated functions based onthe analysis of ftsH double mutants (not shown).

Sensitivity of mutants to a toxic membrane protein hybrid.If the functions that protect cells from tobramycin act by elim-inating mistranslation products that are membrane disruptive,they should also help protect cells from mutant proteins thatare disruptive. To test this possibility, we examined the sensi-tivity of several of the tobramycin-sensitive mutants to theproduction of a MexF-PhoA hybrid protein shown previouslyto be toxic to amgRS mutants (24). Indeed, the toxic mexF-phoA fusion (allele L18) inhibited the growth of both thehtpX-PA5528-yccA mutant and the �amgRS control but hadlittle effect on wild-type growth (Fig. 3). The growth inhibitionwas specifically associated with the production of the toxicMexF-PhoA hybrid protein, since a nontoxic hybrid (allele I17)was innocuous. These findings support the conclusion that one

TABLE 3. Additional proteases and accessory factors contributing to intrinsic tobramycin resistance

Mutationa Function Allele Transposon Position in ORF Readingframe

Tobramycin MICb

(�g/ml)

PAO1 �amgRS

None (MPAO1) 0.5 0.063

Control (PA3303) M7 phoA 312 (392) (�) 0.5 0.063

hslV (PA5053) Protease subunit E12� lacZ 56 (178) �2 0.25 0.031A4� lacZ 74 (178) �3 0.25 0.031G7� lacZ 81 (178) �3 0.25 0.031

hslU (PA5054) Protease subunit B4� lacZ 215 (448) �3 0.25 0.016D2� lacZ 295 (448) �2 0.25 0.031

hflK (PA4942) FtsH protease accessory factor I2� lacZ 43 (401) �1 0.25 0.031I15� phoA 46 (401) �1 0.25 0.031H6� phoA 380 (401) �2 0.25 0.031

hflC (PA4941) FtsH protease accessory factor G4� phoA 55 (290) �2 0.25 0.031C7� lacZ 99 (290) �3 0.125 0.031

a Genes identified in a screen of transposon mutants with insertions affecting predicted proteolytic functions.b Mutations were introduced into PAO1 and the �amgRS mutant using phage lambda Red recombination, and the transposon locations of all of the mutant alleles

were confirmed by resequencing. Tobramycin MICs were determined in duplicate on LB-MOPS agar (with pH adjusted to 7.6 with KOH and no additional salt).

TABLE 4. Sensitivity of ftsH mutants to tobramycin and other treatmentsa

StrainTobramycin MICb (�g/ml) at pH: Growth inhibitory level

7.6 7.0 pHc NaCl (mM)d

Wild type (MPAO1) 0.25 0.5 �9.5 �700�ftsH 0.031 0.063 �9.0 �600�ftsH/Para ftsH��arabinose 0.063 0.125–0.25 �9.25 �600�ftsH/Para ftsH��arabinose 0.25 0.5 �9.5 �700�amgRS 0.031 0.25 �8.0 �200amgR::i63 0.031 0.25 �8.0 �200�ftsH amgR::i63 No growth 0.001 �7.0 �50�ftsH �amgRS No growth 0.001 ND ND

a Growth of 8-�l spots to confluence (�5 � 104 cells) under each condition was scored after 18 h at 37°C. The MIC was the lowest tobramycin concentrationpreventing the lawn growth of the spot of cells. Similarly, the growth inhibitory level was the pH or NaCl concentration range preventing lawn growth. Values areaverages from two replicates.

b Tobramycin sensitivity was measured on LB-MOPS agar (pH 7.0) or LB-EPPS agar (pH 7.6) (32 to 48 mM Na�).c The sensitivity to pH was measured by spotting cells on LB-MOPS (pH 6.75, 7.0, 7.25, and 7.5), LB-EPPS (pH 7.75, 8.0, and 8.25), LB-TAPS (pH 8.5, 8.75, and

9.0), and LB-CAPSO (pH 9.25 and 9.5) agar containing 100 mM Na�.d Sensitivity to NaCl was measured on unbuffered LB agar containing the indicated levels of total NaCl. The salt sensitivity of amgRS mutants was cell concentration

dependent, with reduced sensitivity at low numbers of cells spotted (not shown).

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or more of the three AmgRS-regulated functions contribute tomembrane protein quality control. In contrast, the �ftsH mu-tation did not enhance the growth inhibition due to the toxicmexF-phoA fusion (Fig. 3).

Other phenotypes of tobramycin-sensitive mutants. We nextcharacterized the phenotypes of tobramycin-sensitive mutantsin more detail using phenotype microarray analysis (3). Rela-tive to the parent strain, an amgR mutant exhibited increasedsensitivity to numerous aminoglycosides, several -lactams, al-kaline pH, and NaCl (Fig. 4A and data not shown). Some ofthe phenotypes had been observed previously (24). The htpX-PA5528-yccA triple mutant was hypersensitive to the sameagents but also exhibited increased sensitivity to spectinomy-cin, capreomycin, and several other antibiotics (Fig. 4A anddata not shown). All three mutations of the htpX-PA5528-yccAtriple mutant contributed to the sensitivity phenotypes, sincethe corresponding single and double mutants exhibited smallereffects.

These findings imply that htpX, PA5528, and yccA are largelyresponsible for AmgRS-mediated protection against antibiot-ics, alkaline pH, and sodium chloride. If there are unidentifiedadditional AmgRS-regulated genes that contribute to thesesensitivity phenotypes, then the inactivation of the AmgR reg-ulator in the htpX-PA5528-yccA background should increasethe sensitivities further. To test this possibility, we introducedan amgR mutation into the htpX-PA5528-yccA triple mutantand compared the phenotypes of the triple and quadruplemutants. The quadruple mutant exhibited only slightly in-creased aminoglycoside and alkaline pH susceptibilities (Fig. 4and data not shown), indicating that HtpX, PA5528, and YccA

FIG. 3. Toxicity of a MexF-PhoA hybrid protein toward tobramy-cin-sensitive mutants. The effect of the expression of a toxic (alleleL18) or nontoxic (allele I17) MexF-PhoA hybrid on the growth ofmutants is shown. Toxicity was assayed by growing cultures underpermissive conditions (in LB-MES [pH 6.0]), followed by spotting10-fold dilutions on LB-MOPS (pH 7.7) agar containing 86 mM Na�.

FIG. 4. Phenotypic analysis of tobramycin-sensitive mutants. Antibiotic sensitivities (A) and pH and NaCl sensitivities (B) of mutants werecharacterized using phenotype microarray analysis. Each panel shows the tetrazolium reduction during 48 h, a measure of respiration. Tetrazoliumreduction by each mutant (green) is compared to that of a control strain (red), with overlapping regions in yellow. Tested mutants were comparedto the PA3303 insertion control, with the exception of �ftsH, which was compared to MPAO1. For antibiotics, growth across four progressivelyhigher concentrations is presented from left to right. The following mutants were tested (see Table S1 in the supplemental material): AHP36,AHP126, AHP83, AHP124, AHP125, AHP147, AHP146, AHP145, AHP127, AHP241, and SLP1742.

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primarily are responsible for these resistances. In contrast, thequadruple mutant was significantly more sensitive than thetriple mutant to NaCl (Fig. 4B), indicating that additionalunidentified AmgRS-regulated functions contribute to salt re-sistance.

The ftsH deletion mutant exhibited phenotypes similar tothose of the amgR mutant (Fig. 4 and Table 4). The resultsfurther support the conclusion that the two functions providepartially overlapping protective functions.

DISCUSSION

We report a genetic dissection of aminoglycoside intrinsicresistance in P. aeruginosa. The functions we identified definea functional network underlying resistance in which proteolysisplays a central role (Fig. 5) and in which the membrane pro-tease FtsH is particularly important. Our results further sup-port models in which membrane-disruptive proteins resultingfrom misreading promote aminoglycoside action and suggeststhat proteolysis protects cells from such proteins (6, 24).

Previously, we showed that P. aeruginosa intrinsic aminogly-coside resistance requires the two-component regulator AmgRS(24). While its precise role was unclear, insertions in amgRSled to the greatest increase in tobramycin sensitivity observedin a comprehensive mutant screen. We now show that AmgRScontrols three genes, the combined action of which largelyaccounts for its role in resistance. The first, yccA, encodes aprotein whose E. coli homologue modulates the activity ofmembrane protease FtsH. The second, htpX, encodes a mem-brane protease itself. The third, PA5528, encodes a membraneprotein of unknown function.

Because two of the three AmgRS-regulated resistance func-tions are associated with proteolysis, we next examined theconsequences of inactivating 66 predicted proteases and re-lated factors. This focused screen confirmed previous findingsthat two additional FtsH regulators (HflC and HflK) and acytoplasmic protease (HslVU) contribute modestly to intrinsictobramycin resistance.

Finally, we examined the effect of inactivating FtsH pro-tease. Although FtsH is essential under standard growth con-ditions, we isolated an ftsH deletion mutant using a permissive

medium. The deletion of ftsH leads to a striking increase intobramycin sensitivity, about the same as that of eliminatingamgRS.

The physiological role of FtsH in P. aeruginosa has not beencharacterized. However, in E. coli, FtsH and its accessory fac-tors are largely responsible for membrane protein quality con-trol (18). FtsH exists in an integral cytoplasmic membranecomplex that includes HflC and HflK, which regulate its spec-ificity (21, 22). FtsH activity also is controlled by YccA, whichinhibits the FtsH-mediated degradation of SecY “jammed” bypolypeptides it cannot export (34). The positive role of bothYccA and FtsH in P. aeruginosa aminoglycoside resistanceseems paradoxical, since in E. coli YccA antagonizes ratherthan stimulates FtsH activity (1, 34). However, it is not clearthat the YccA contribution to tobramycin resistance is medi-ated via FtsH.

The two other proteases that contribute to intrinsic amino-glycoside resistance in P. aeruginosa are HtpX and HslVU.HtpX is a cytoplasmic membrane protease whose function inE. coli appears to overlap with FtsH, since double mutantslacking both proteases exhibit a synthetic growth defect (32).HslVU is a cytoplasmic ATP-dependent protease distantly re-lated to the eukaryotic proteasome (15, 20). HtpX and HslVUmay perform redundant back-up functions for FtsH in P.aeruginosa or act on disruptive polypeptides FtsH does notrecognize. The participation of soluble as well as membraneproteases in aminoglycoside resistance indicates that some dis-ruptive polypeptides are degraded in the cytoplasm.

For several of the resistance functions we examined, therewas enhanced sensitivity of multiple mutants relative to that ofsingle-mutant parents. Such additive effects imply that the cor-responding functions act independently. For example, the to-bramycin MICs for amgR and ftsH single mutants were 8- to16-fold lower than the MIC for the wild type, whereas theamgR-ftsH double mutant was 500-fold more sensitive. Muta-tions in the three protective functions whose expression isregulated by AmgRS (YccA, HtpX, and PA5528) also showedadditive effects.

If the resistance functions we identified truly participate inmembrane protein quality control, their inactivation shouldrender cells sensitive to aberrant membrane proteins. To testthis prediction, we examined mutant sensitivity to a toxic mem-brane protein hybrid (MexF-PhoA) identified in previous stud-ies (24). As expected, the inactivation of AmgRS-regulatedfunctions increased sensitivity to the aberrant membrane pro-tein. Unexpectedly, FtsH inactivation did not appear to affectsensitivity, indicating that the AmgRS response is more impor-tant in eliminating some classes of toxic proteins.

If membrane protein quality control is important to bacteriaunder normal growth conditions, mutations compromising itshould alter traits other than aminoglycoside susceptibility.Indeed, we found that enhanced sensitivity to several classes ofantibiotics, alkaline pH, NaCl, and other substances trackedclosely with aminoglycoside sensitivity. We suspect that thesephenotypes result from a compromised inner membrane dueto spontaneous (rather than aminoglycoside-stimulated) mis-translation products. The compromised bilayer may be unableto sustain the increased transmembrane potential necessary tomaintain the proton-motive force at elevated pH and also may

FIG. 5. Functions responsible for intrinsic aminoglycoside resis-tance. Relationships among the genes contributing to tobramycin re-sistance are presented. For clarity, the MexXY-OprM efflux pump,MexF-PhoA, and other resistance factors are not included.

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allow the greater internalization of sodium ions and other toxicsubstances.

This analysis has helped define a network of functions re-sponsible for aminoglycoside intrinsic resistance (12, 24). Ourresults strongly support models proposing that resistance inlarge part reflects quality control mechanisms that protect cellsfrom mistranslation products. Such protective functions repre-sent novel targets for drugs that could enhance the clinicalefficacy of aminoglycosides.

ACKNOWLEDGMENTS

We thank Larry Gallagher, Day Hills, and Pradeep Singh for helpfulcomments.

This work was supported by grants from the Cystic Fibrosis Foun-dation (MANOIL08G0) and the National Institutes of Health(R21AI078495).

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