Genetic Analysis of 15 Protein Folding Factors and ... · prefers unfolded proteins with...

Genetic Analysis of 15 Protein Folding Factors and Proteases of theEscherichia coli Cell Envelope

Juliane Weski and Michael Ehrmann

Centre for Medical Biotechnology, Faculty of Biology and Geography, University Duisburg-Essen, Essen, Germany

Each cell hosts thousands of proteins that vary greatly in abundance, structure, and chemical properties. To ensure that all pro-teins are biologically active and properly localized, efficient quality control systems have evolved. While the structure, function,and regulation of some individual protein folding factors and proteases were resolved up to atomic resolution, others remainpoorly characterized. In addition, little is known about which factors are required for viability under specific stress conditions.We therefore determined the physiological implications of 15 factors of the E. coli cell envelope by an integrated genetic ap-proach comprising phenotypic analyses. Our data indicate that surA and tsp null mutations are a lethal combination in rich me-dium, that surA dsbA and surA dsbC double mutants are temperature sensitive, and that surA ptrA, surA yfgC, dsbA fkpA, degPtsp, degP ppiD, tsp ppiD, and degP dsbA double mutants are temperature sensitive in rich medium containing 0.5 M NaCl, whiledegP dsbA, degP yfgC, tsp ydgD, and degP tsp double mutants do not grow in the presence of SDS/EDTA. Furthermore, we showthat in degP dsbA, degP tsp, and degP yfgC double mutants a subpopulation of LamB exists as unfolded monomers. In addition,dsbA null mutants expressed lower levels of the outer membrane proteins LptD, LamB, FhuA, and OmpW while FhuA levelswere reduced in surA single and degP ppiD double mutants. Lower FhuA levels in degP ppiD strains depend on Tsp, since in a tspdegP ppiD triple mutant FhuA levels are restored.

The cell envelope of the Gram-negative bacterium Escherichiacoli represents an experimental model system to address the

physiological implications of protein quality control involvingprotein diagnosis, repair, and turnover (45). Protein quality con-trol systems are mainly comprised of unfolded protein responsesignal transduction pathways, molecular chaperones, folding cat-alysts, and proteases. Chaperones recognize exposed hydropho-bicity of proteins present in a nonnative state, thereby discrimi-nating against native proteins. Two factors, Skp and Spy, aregeneral molecular chaperones in the E. coli periplasm. Skp func-tion has so far been mainly attributed to the folding and assemblyof outer membrane proteins (49), and Spy prevents protein aggre-gation and promotes protein folding (54).

Protein folding catalysts carry out related functions, but incontrast to chaperones they improve slow steps in protein foldingby catalyzing disulfide (S-S) bond formation or cis-trans isomer-ization of proline residues. The periplasmic redox machinery isgenetically and biochemically well studied. The main players be-long to the Dsb family, which are involved in the formation(DsbA), reduction (DsbC), and isomerization (DsbC and DsbA)of S-S bonds (31, 41). Native substrates include dozens of proteinssuch as alkaline phosphatase, OmpA, heat-stable enterotoxins,LamB, periplasmic binding proteins, pilin proteins, and compo-nents of various secretion apparatuses involved in virulence.There are also four proline isomerases in the cell envelope, PpiA,SurA, PpiD, and FkpA. While an involvement in outer membranebiogenesis has been described for some proline isomerases, theirexact physiological implications remain to be determined (55).

Proteases promote protein turnover by catalyzing the cleavageof peptide bonds. There are at least 24 proteolytic enzymes in thecell envelope of E. coli. While a few are well studied, many haveonly been identified via bioinformatics (36). For example, DegPand DegQ are members of the widely conserved HtrA family ofserine proteases that are implicated in the tolerance against vari-ous folding stresses, including bacterial pathogenicity (16, 17).

These functions are based on unique features such as the combi-nation of chaperone and protease activities and the reversibleswitch from the resting hexameric to the active dodecameric ortetracosameric conformations (2, 29, 35, 62, 66). This switch isbased on an allosteric mechanism that involves binding of mis-folded proteins to the PDZ domain and the active site (34, 44, 46,62). Interestingly, 12- and 24-meric DegP cages contain foldedmonomers of mislocalized porins, implicating DegP in the bio-genesis of outer membrane proteins (35). There are other factorsin the cell envelope that are bifunctional. For example, chap-erone activity has been demonstrated for DegQ and the prolineisomerase SurA, as well as for the redox factors DsbC and DsbG(7, 15, 40, 65).

Other proteases such as the serine protease Tsp and the metal-loprotease PtrA (pitrilysin A) have been implicated in proteinquality control. For example, a Tn5 insertion in tsp increases thesensitivity to multiple antibiotics, probably because of a defectiveouter membrane (64), and degQ is a multicopy suppressor of tspnull mutations (5). In addition, like other PDZ proteases, Tspprefers unfolded proteins with hydrophobic C termini as sub-strates (6, 67, 72). Even though less information on PtrA is avail-able, the degradation of misfolded maltose binding protein mu-tant 31 (8) and �-lactamase (3) suggest a role in protein qualitycontrol.

There are four hypothetical proteases in the cell envelope thatmight be involved in protein quality control. YdgD is a small

Received 13 February 2012 Accepted 5 April 2012

Published ahead of print 13 April 2012

Address correspondence to Michael Ehrmann, [email protected].

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

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

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periplasmic serine protease. It is expressed in tissues of a chickeninfection model, and ydgD mutants exhibit increased sensitivitytoward the antibiotics ampicillin and cephradine, which target cellwall biosynthesis (19, 38). The predicted metalloproteases YfgC,YcaL, and YggG are homologous proteins belonging to the widelyconserved M48 family (56), prominent members of which includethe mammalian OMA1 and STE24 proteases (4, 42). The yfgCpromoter is regulated by sigma E, i.e., protein folding stress (23,57), and yfgC mutants are hypersensitive toward multiple antibi-otics (24, 38). The other M48 family members, YcaL and YggG, areouter membrane lipoproteins (37, 69). The yggG promoter is up-regulated by heat shock (28) and by the Rcs two-component sys-tem (25), while insertions within the ycaL gene are lethal in Sal-monella enterica (32) but not in E. coli.

Like all other cells, E. coli has developed compartment-specificsystems to respond to the presence of misfolded proteins. In thecell envelope, two major regulons have been identified, Sigma E(RpoE) and Cpx (60). Targets of the Sigma E pathway include thedegP and fkpA promoters and rpoH, encoding the cytoplasmicheat shock sigma factor, as well as rpoE and its regulators rseABC(55). Among the known protein quality control factors that areregulated by two-component Cpx pathway are the promoters ofthe degP, dsbA, ppiD, ppiA, and spy genes (55).

To obtain information about the physiological implications ofestablished and hypothetical periplasmic protein quality controlfactors, we analyzed 15 genes by studying the synthetic pheno-types of combined knockout mutations, membrane integrity,metabolomic profiling, induction of stress response genes, and thebiogenesis of outer membrane proteins.

MATERIALS AND METHODSBacterial strains, plasmids, and culture conditions. Strains containingnull mutations were obtained from the E. coli Genetic Stock Centre, whichdistributes the Keio collection (1). These mutations were introduced intowild-type (wt) strain BW30270 (E. coli Stock Center no. 7925) by P1transduction (47) to generate isogenic strains containing the individualand double mutants (see Table S1 in the supplemental material). To verifythe constructed strains, Western blot assays or PCR analyses were per-formed.

Bacterial cultures were routinely grown at 37°C in rich medium con-taining 10 g/liter NZ-amine A, 5 g/liter Bacto yeast extract, and 10 g/literNaCl. When required, antibiotics were added to the following final con-centrations: ampicillin, 200 �g/ml; kanamycin, 100 �g/ml; tetracycline, 5�g/ml; and chloramphenicol, 15 �g/ml.

Growth tests. Bacterial cultures were grown at 37°C in rich mediumovernight. Cells (103) were spotted onto rich medium agar plates contain-ing either 0.5% SDS and 0.5 mM EDTA or 500 mM NaCl. Plates wereincubated at various temperatures.

Assay of membrane permeability: �-galactosidase assays. Mem-brane defects were determined by assaying levels of �-galactosidase in thesupernatant of the growth medium. �-Galactosidase assays were per-formed as described previously (20), except using the supernatant insteadof cell lysate. Cells were grown in rich medium, and the wt lac operon wasinduced with 50 �M IPTG (isopropyl-�-D-thiogalactopyranoside) andgrown further until mid-log phase. Cells (5 � 108) were harvested bycentrifugation (4,000 � g, 10 min, 4°C), and 50 �l of the supernatant wasmixed with 100 �l ONPG (o-nitrophenyl-�-D-galactopyranoside) (4 mg/ml). �-Galactosidase activities are expressed as �(OD420/t)/(OD600 � V),where OD420 and OD600 are the optical densities at 420 and 600 nm,respectively, and V refers to the volume of supernatant of the culture.

Analyses of outer membrane proteins. Outer membrane fractionswere isolated from cells grown in rich medium until mid-log phase. Cellswere harvested by centrifugation, washed with 100 mM NaCl, and resus-

pended in 10 mM HEPES, pH 7.4. After DNase digestion, cells were bro-ken and cell debris was removed by centrifugation (10 min, 16,000 � g).Membrane fractions were pelleted by an additional centrifugation step(45 min, 120,000 � g). The pellet was resuspended in buffer (10 mMHEPES, pH 7.4, 2% [wt/vol] lauryl sarcosine) and stirred overnight at4°C. The outer membrane fraction was pelleted by ultracentrifugation(45 min, 120,000 � g), washed twice, and resuspended in 10 mMHEPES, pH 7.4.

For detection of unfolded LamB monomer, cells were grown untilmid-log phase in liquid rich medium at 37°C and gentle lysis was per-formed (69a).

Phenotype microarray assays. Phenotype microarrays were per-formed at Biolog, Inc., Hayward, CA, as described previously (9).

RNA extraction and real-time quantitative PCR (RT-qPCR). For ex-traction of total RNA, cells were grown in M9 minimal medium untilmid-log phase (47). RNA was stabilized, isolated, and DNase digestedusing the RNeasy kit (Qiagen) according to the manufacturer’s protocol.RNA (1 �g) was used for cDNA synthesis using the First Strand cDNAsynthesis kit (Fermentas). cDNA (45 ng) was quantified on Rotor-Gene3000 (Corbett Research) by using Absolute QPCR SYBR green mix(Thermo). After an initial 15-min activation step at 94°C, 45 cycles (94°Cfor 15 s, 56°C for 30 s, 72°C for 30 s, 78°C for 15 s, and 84°C for 15 s) wereperformed. A melting curve analysis was performed at the end of cyclingto ensure that there was single amplification. Transcript levels were nor-malized to the level of gapA, and quantitation analysis was performed asdescribed previously (51). Assays were done with two biological and threestatistical replicates, and standard deviations were calculated.

RESULTSIdentification of synthetic phenotypes of double-null mutants.To obtain in vivo evidence for the functional importance of pro-tein quality control factors of the E. coli cell envelope, we con-structed 113 isogenic derivatives of wt strain BW30270, i.e., 15single mutants and 98 double mutants. The single-knockout mu-tants were those with deletions of the ppiA, surA, ppiD, fkpA, dsbA,dsbC, and skp genes, encoding folding factors, and deletions of thedegP, degQ, ptrA, tsp, ydgD, yfgC, ycaL, and yggG genes, encodingproteases. Of those, double mutant strains were constructed in themost possible combinations, except that a dsbA dsbC deletionstrain was not constructed because the biology of the disulfidebond formation system is well understood (31), nor were strainswith double deletions of the ppiA, surA, ppiD, and fkpA genesconstructed, because previous work suggested that the pheno-types of the quadruple-null mutant were very similar to those ofthe individual surA null mutant (30). The constructed strains wereused to test growth on rich medium plates at 28, 37, and 43°C.Phenotype analyses verified published evidence, i.e., that surAdegP and surA skp double mutations are synthetic lethal combina-tions (58). In addition, surA tsp double mutations were found tobe synthetically lethal (Table 1). In addition, dsbA surA and dsbCsurA double mutants were inviable at 43°C and exhibited reducedgrowth at 37°C. Table 1 lists only those double mutants that haveadditional phenotypes compared to the corresponding single-nullmutants. A complete list of all mutants generated and their phe-notypes is provided in Table S2 in the supplemental material.

Stress conditions. We subsequently tested growth of the singleand double mutants under conditions causing protein foldingproblems, including hyperosmolarity and sensitivity toward SDS/EDTA in combination with elevated temperatures. On plates con-taining 500 mM NaCl (Table 1), surA, dsbA, and tsp single mutantsshowed reduced growth at 37°C, indicating that these factors areindividually important for growth under salt stress. In addition,

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the ppiD degP double mutant showed reduced growth at 37°C. At42°C, surA, dsbA, and tsp single mutants displayed severely re-duced growth. Of the double-null mutations, surA ptrA, surAyfgC, tsp ppiD, degP ppiD, degP tsp, dsbA degP, and dsbA fkpA weresynthetic lethal combinations.

Media containing SDS and EDTA are commonly used to detectdefective cell envelopes (7). Therefore, strains were grown on rich-medium plates containing 0.5% SDS and 0.5 mM EDTA at 37 and42°C. Under these conditions, surA single mutants and their cor-responding double mutants were inviable. degP mutants showedreduced growth at 37°C and no growth at 42°C. In addition, dsbA,ppiD, and tsp single mutants showed reduced growth at 37 and42°C. Furthermore, dsbA degP, tsp degP, yfgC degP, and ydgD tspwere synthetic lethal combinations at 37°C and ydgD tsp was syn-thetically lethal at 42°C (Table 1). Since dsbA degP and tsp degPwere also lethal combinations when cells were grown in hyperos-molar media at 42°C, these data suggest that DsbA, DegP, and Tspcontribute to survival under stress conditions.

Metabolic profiling. To extend the phenotypic analyses, globalmetabolic profiling was performed using phenotype microarraysexamining 1,920 different growth conditions for each mutantstrain (9). Based on the data generated so far, we selected a set of 10single mutants (surA, fkpA, ppiD, dsbA, dsbC, degP, tsp, ydgD, yfgC,and ptrA mutants) and 12 double mutants (dsbA surA, dsbC surA,ptrA surA, yfgC surA, dsbA degP, tsp degP, yfgC degP, ppiD degP,ydgD tsp, ppiD tsp, fkpA tsp, and dsbA fkpA mutants) for pheno-type microarray analyses that were performed at 37°C (Fig. 1; seealso Fig. S1 in the supplemental material). Among the single mu-tants, the tsp mutant exhibited the most severe phenotypes. Thetsp mutant was particularly sensitive to pH and chemicals (anti-microbials and dyes) and somewhat sensitive to medium osmo-larity and ions. This phenotype is qualitatively similar to pheno-type microarray data obtained for degP mutants grown at 42°C.However, at 37°C, a degP mutant displayed almost no significantphenotypes. The surA single mutant was hypersensitive to KCl,NaCl, and Na2SO4 as well as to folate antagonists, macrolides, andrifamycin/rifampin. The macrolide hypersensitivity is also ob-served in the yfgC mutant. The dsbA mutant displayed scatteredphenotypes.

Of the double mutants, the surA dsbA mutant had the strongestphenotypes, as most growth conditions were negatively affected,confirming the original observation that this double mutant issynthetically lethal on rich medium plates at 42°C and severelycompromised at 37°C (Table 1). While the individual dsbA anddegP mutants had only a few phenotypes, the double mutant dis-played a severe hypersensitivity toward chemicals, pH, and ions. Asimilar although even stronger phenotype was observed for the tspdegP double-null strain. In addition to the hypersensitivity againstchemicals, pH, and ions, this strain was unable to use many car-bon sources and peptides as nitrogen sources. A rather unusualphenotype was observed for ptrA surA and yfgC surA double mu-tants. These strains did not grow in minimal media (Fig. 1), exceptwhen leucine was added to the growth medium. The reason for theLeu auxotrophy is unknown. In addition, ppiD tsp and fkpA tspdouble mutants exhibited fewer phenotypes than did the tsp singlemutant (see Fig. S1 in the supplemental material), suggesting thatppiD and fkpA null mutants are suppressors of the tsp null mutantunder some but not all growth conditions. Together, these in vivoresults confirm the physiological importance of the folding factorsand proteases investigated.

Membrane permeability. Since folding factor and proteasemutations can cause defects in integral membrane protein biogen-esis and are known, for example, to increase sensitivity to antibi-otics due to increased membrane permeability, we tested the in-tegrity of the cytoplasmic and outer membranes of mutants thatdisplayed growth defects or synthetic lethality in the assays de-scribed above by measuring the release of cytoplasmic �-galacto-sidase into the growth medium from cells grown in rich mediumat 37°C. Since �-galactosidase is a homotetramer of �450 kDa, itsrelease from cells indicates severe membrane defects that couldresult in cell lysis. About 4% of the expressed �-galactosidase wasreleased into the growth medium in the surA single mutant, whiletsp, degP, and dsbA single mutants leaked 4- to 10-fold less �-ga-lactosidase into the growth medium than did the surA mutant.Two double mutants, degP tsp and degP dsbA, leaked twice asmuch and as much �-galactosidase, respectively, into the mediumas the surA mutant. tsp ydgD and tsp fkpA mutants leaked aboutone-half the amount of �-galactosidase that the surA single mu-

TABLE 1 Growth of cells lacking various folding factors and proteasesunder different conditionsa

Strain orrelevantgenotype

Growth of cells under indicated condition(s)

Temp (°C) 0.5 mM NaCl SDS/EDTA

28 37 43 37°C 42°C 37°C 42°C

BW30270 (wt) �� surA �� ppiD �� fkpA �� ppiA �� skp �� dsbA �� dsbC �� degP �� degQ �� tsp �� ptrA �� yfgC �� ydgD �� ycaL �� yggG �� surA degP ND ND ND ND ND ND NDsurA skp ND ND ND ND ND ND NDsurA tsp ND ND ND ND ND ND NDsurA ptrA �� surA yfgC �� surA dsbA �� surA dsbC �� degP yfgC �� tsp ydgD �� tsp ppiD �� degP ppiD �� degP tsp �� dsbA degP �� dsbA fkpA �� a Sizes of cell colonies were determined following growth on (i) rich-medium agarplates, (ii) hyperosmolar rich-medium agar plates containing 500 mM NaCl, and (iii)rich-medium agar plates containing 0.5% SDS plus 0.5 mM EDTA following overnightincubation at the temperatures indicated. ��, growth comparable to that of wtstrain BW30270; ��, weak growth; �, minimal growth; �, no growth; ND, not donebecause these mutations were lethal combinations. The phenotypes of all strainsconstructed are provided in Table S2 in the supplemental material.

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tant leaked, while degP yfgC and dsbA fkpA double mutants leakedat least twice as much �-galactosidase as the corresponding indi-vidual single mutants (Table 2). Leakage of �-galactosidase intothe growth medium could result from cell lysis, even though oth-ers reported membrane permeability (leakage of DNA and pro-teins) without loss of viability (33).

Induction of protein stress response genes. Since folding fac-tors and proteases perform important tasks in protein quality con-trol, their absence is expected to induce promoters of genes thatare controlled by unfolded protein response pathways. We there-fore tested the induction of the stress genes rpoE and spy, which aremembers of the sigma E and Cpx regulons and the Cpx and Baeregulons, respectively (50, 52) in surA dsbC, dsbA fkpA, degP dsbA,degP ppiD, degP yfgC, tsp fkpA, tsp ppiD, degP tsp, and tsp ydgDdouble mutants and the corresponding single mutants. These mu-tations were chosen because of their phenotypes in the previousassays. All other surA double mutants could not be investigatedbecause they did not grow in minimal medium. Differential ex-pression of the rpoE and spy genes was measured by RT-qPCRafter growth of cells in minimal media at 37°C (Table 3).

The strongest induction of rpoE was observed in surA mutants(factor of 5), confirming earlier observations (7). This effect canbe explained by the fact that SurA is involved in the biogenesis ofouter membrane proteins and mislocalized outer membrane pro-

teins are the main inducers of the sigma E pathway (60). In addi-tion, the single dsbA null allele (2.6-fold induction) as well as thedsbA fkpA (3.3-fold induction), degP dsbA (3.5-fold induction),and degP tsp (2.3-fold induction) double-null mutations inducedrpoE expression. Interestingly, these factors are also involved inouter membrane protein biogenesis (Fig. 2 and 3). Moreover, rpoEexpression was induced 2.3-fold in tsp ydgD cells, while the com-bination of surA and dsbC null mutations decreased rpoE pro-moter activation compared to that of the single surA mutant from5- to 3.2-fold. This slightly weaker phenotype was also observedwhen assaying membrane permeability (Table 2).

The strongest induction of the spy gene was observed for degPtsp (12.9-fold induction) and for dsbA fkpA (11.2-fold induction)mutants. Other strong inducers of spy included the degP dsbA(7.7-fold induction), dsbA (6-fold induction), surA dsbC (6-foldinduction), tsp ydgD (4.3-fold induction), tsp ppiD (4.9-fold in-duction), and tsp fkpA (2.5-fold induction) mutations, while thesurA single-null mutation induced spy expression by a factor of 2.8(Table 3).

In general, mutations that caused a strong induction of eitherrpoE or spy also induced the other stress gene as well. The onlyexception was surA dsbC, causing an activation of spy but a slightreduction of rpoE expression compared to the surA single muta-tion. In addition, the results of these tests correlate with thephenotypes detected for the double mutants dsbA fkpA (salt

FIG 1 Global metabolic profiling. Phenotype microarrays of selected knockout mutants (comparison of wt and mutant strains) grown at 37°C unless otherwiseindicated. Yellow indicates metabolic activity of the mutant comparable to that of the wt strain, green indicates better metabolic activity than wt, and red indicatesreduced metabolic activity compared to wt. In each panel of 20 rectangles, each rectangle represents a 96-well plate subjected to various growth conditions. Toprow (from the left): the first two plates show carbon sources, followed by N, P, S, and nutrient supplements. Second row from top: the first three plates show Nsources followed by osmotic compounds and ions (2 plates). In the lower two rows, the sensitivity to chemicals (detergents, antibiotics, dyes, etc.) is tested. Boxesindicate wells where the metabolic activity of the mutant is significantly different from that of the wt. The complete results of all phenotype microarrayexperiments are provided in Fig. S1 in the supplemental material.

TABLE 2 Leakage of �-galactosidase into the medium of cells lackingfolding factorsa

Strain or relevant genotype�-Galactosidase activity(�mol/min)

Strain BW30270 (wt) �1surA 43ppiD �1fkpA 1dsbA 4dsbC �1tsp 9degP 5ptrA 1yfgC 2ydgD 1dsbA fkpA 10degP dsbA 41degP yfgC 13degP tsp 81surA dsbC 24tsp fkpA 23tsp ydgD 24tsp ppiD 7a Mutant strains were grown to mid-log phase in liquid rich medium at 37°C. Activityof cytoplasmic �-galactosidase in cell culture supernatant was determined as anindicator of a membrane defect. Assays were done with two biological and threestatistical replicates, and standard deviation was �40%. Total �-galactosidase activity ofcells was on average 970 �mol/min.

TABLE 3 Induction of rpoE and spy genes in folding factor mutantsa

Strain or relevant genotype

Fold induction (meannormalized expression) of:

rpoE spy

BW30270 (wt) 1.0 1.0surA 5.0 2.8dsbA 2.6 6.0degP 1.1 0.9tsp 1.3 1.4dsbC 1.4 1.0fkpA 1.2 1.3ppiD 1.2 1.0ydgD 0.7 0.9yfgC 1.0 0.9surA dsbC 3.2 6.0dsbA fkpA 3.3 11.2degP dsbA 3.5 7.7degP tsp 2.3 12.9tsp fkpA 1.1 2.5tsp ydgD 2.3 4.3tsp ppiD 1.8 4.9degP ppiD 0.8 0.9degP yfgC 1.2 1.1a Cells were grown to mid-log phase in liquid minimal medium at 37°C. RNA levels ofrpoE and spy were determined by RT-qPCR. Assays were done with two biological andthree statistical replicates, and standard deviations were �40%.




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stress and membrane permeability), degP dsbA (salt and EDTA/SDS stress and membrane permeability), degP tsp (salt andEDTA/SDS stress and membrane permeability), and tsp ydgD(EDTA/SDS stress and membrane permeability) (Table 1).

Implications of folding factors and proteases in the biogene-sis of outer membrane proteins. Having so far performed globalin vivo analyses, we determined the fates of individual proteins inselected single and double mutants (Fig. 2 and 3). We focused onouter membrane proteins because these proteins have been usedpreviously to characterize, for example, surA, degP, and skp mu-tants (68). First, we isolated outer membrane fractions and deter-mined the abundance of FhuA, Imp (LptD), LamB, OmpA,OmpC, OmpF, OmpW, and PhoE by Western blotting. In thefollowing, we describe only pronounced effects (marked by redboxes in Fig. 2A). We detected that OmpW biogenesis is severelyaffected in surA single mutants and consequently in the relevantdouble mutants. OmpW is a conserved monomeric porin formingan 8-stranded � barrel (27). FhuA levels were strongly reduced indegP ppiD double mutants. FhuA, a monomeric porin forming a22-stranded � barrel, is the receptor for ferrichrome-iron, for theantibiotic albomycin, for several bacteriophages, and for the bac-terial toxin colicin M (13, 21). Furthermore, dsbA mutants pro-duced reduced levels of FhuA and of the lipopolysaccharide (LPS)

transporter Imp (LptD) (22), the maltoporin LamB (63), andOmpW. These proteins have 4, 4, 2, and 0 Cys residues, respec-tively, and S-S bonds were described for FhuA, Imp (LptD), andLamB (10, 12, 39). We were also able to reproduce the previouslyreported phenotypes of the surA mutant, i.e., lower levels of Imp(LptD), FhuA, and LamB (68, 71). These reductions were there-fore also seen when the surA mutation was combined with dsbA,dsbC, ptrA, and yfgC mutations. Surprisingly, however, FhuA lev-els were not reduced in the surA dsbA double mutant. The reasonfor this effect is unknown, and it cannot be explained by the otherphenotypes detected so far. Since Imp (LptD) levels are reduced indsbA and surA mutants (Fig. 2A), these strains will have reducedLPS levels. Reduced LPS levels will interfere with the biogenesis ofouter membrane proteins, the assembly of which depends on LPSsuch as OmpA, PhoE, and presumably FhuA (14, 18, 21). In ad-dition, we observed other published effects, i.e., reduced levels ofLamB in surA and dsbA single mutants and of OmpC and OmpF insurA mutants in outer membrane preparations (data not shown)(53, 59, 68, 71).

Since the expression levels of genes encoding outer membraneproteins can be affected in folding factor mutants (71), we deter-mined the mRNA levels in those strains exhibiting defects in outermembrane protein biogenesis (Fig. 2B). Reduced mRNA levelswere observed for lamB and ompW in surA mutants as well as forlamB in degP ppiD double mutants. Therefore, the effects of surAon the levels of LamB and OmpW and the small effect of degP ppiDon LamB levels were most likely indirect. The effects of surA onImp/LptD and FhuA biogenesis were previously reported to bedirect, as mRNA levels were not affected (71). Therefore, proteinlevels of LamB, FhuA, and OmpW depend on the presence ofDsbA, and those of FhuA depend on the presence of DegP, PpiD,and SurA. Furthermore, since FhuA protein levels were reduced indegP ppiD double mutants, we tested whether FhuA is degraded byTsp under these conditions. Western blotting confirmed this hy-pothesis, i.e., FhuA levels are increased in degP ppiD tsp triplemutants compared to the degP ppiD double mutants (Fig. 2C).

In a second set of experiments, we analyzed the levels of LamB

FIG 2 Outer membrane protein biogenesis. (A) Steady-state levels of various proteins in the outer membrane fraction of indicated mutant strains. Pronouncedeffects are marked by red boxes. Braun’s outer membrane lipoprotein (Lpp) served as a loading control. (B) mRNA levels of fhuA, lamB, and ompW werecompared in wt and mutant strains by RT-qPCR as described in Table 3. Standard deviation was below 40%. For comparative and quantitative analysis, transcriptlevels were normalized to the level of gapA. (C) FhuA levels in various mutant strains. The comparison of FhuA levels in degP ppiD mutants with those in degPppiD tsp mutants suggests that Tsp degrades FhuA in degP ppiD strains.

FIG 3 LamB folding intermediates in various mutant strains. Bacteria weregently lysed and incubated at 37°C for 10 min (A) or heated at 95°C for 5 min(B). The apparent molecular mass is 80 kDa for trimeric LamB and 49 kDa forthe unfolded monomer. Glucose-6-P-dehydrogenase served as a loading con-trol (C).

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in whole-cell lysates to detect the presence of unfolded monomersin the periplasm (Fig. 3). Significant amounts of unfolded mono-mers were detected in degP dsbA and degP tsp strains, while tracesof unfolded monomers were observed in degP yfgC strains.

DISCUSSION

A systematic genetic approach involving 15 conserved genes wasused to determine the phenotypes of single and double mutants byassaying 7 growth conditions (Table 1), membrane integrity (Ta-ble 2), and induction of unfolded protein response genes (Table 3)and by metabolomic profiling (Fig. 1). These results reveal a rolefor the to-date poorly characterized periplasmic proteases YfgCand YdgD in protein quality control (see the phenotypes of thecorresponding mutants listed below). While our data confirm thesynthetic lethal phenotypes of surA degP and surA skp mutations,surA tsp was identified as an additional lethal combination. Fur-thermore, 14 synthetic lethal phenotypes were detected under spe-cific growth conditions: surA dsbA (43°C), surA dsbC (43°C), surAptrA (0.5 M NaCl at 42°C), surA yfgC (0.5 M NaCl at 42°C), dsbAfkpA (0.5 M NaCl at 42°C), degP dsbA (0.5 M NaCl at 42°C andSDS/EDTA at 37°C), degP yfgC (SDS/EDTA at 37°C), degP tsp (0.5M NaCl at 42°C and SDS/EDTA at 37°C), degP ppiD (0.5 M NaClat 42°C), tsp ppiD (0.5 M NaCl at 42°C), and tsp ydgD (SDS/EDTAat 37°C and 42°C) double mutants (Table 1). Future studies arerequired to understand the reasons for these phenotypes. How-ever, since dsbA fkpA, degP dsbA, degP yfgC, degP tsp, and tsp ydgDmutants release �-galactosidase into the growth medium, pre-sumably because of cell lysis (Table 2), a membrane defect couldbe responsible for the synthetic lethality under the stress condi-tions described above.

The graphic representation of the detected negative syntheticphenotypes (Fig. 4) illustrates that the combinatory effects of sin-gle mutations vary significantly depending on the growth (stress)conditions. These results suggest that under each specific stresscondition tested, a specific set of protein quality control factors iscritical for cell fate. Furthermore, the relative importance of indi-vidual factors can be deduced by simply counting the number ofsynthetic phenotypes for each gene. Based on these criteria, surA(7 negative synthetic combinations with other single mutations),degP (5 such combinations), tsp (5 combinations), and dsbA (3combinations) appear to be more important than yfgC (2 combi-nations), fkpA (2 combinations), and dsbC, ptrA, skp, and ydgD (1combination) or degQ, ppiA, ycaL, and yggG (none).

Metabolomic analyses monitoring 1,920 growth conditions ina single experiment revealed the global importance of cell enve-lope folding factors for cell growth (Fig. 1). For example, tsp singleand degP dsbA and surA dsbA double mutants are highly sensitiveto chemicals, including antibiotics and dyes, confirming earlierreports for tsp (64). Also, the surA yfgC and surA ptrA doublemutants are leucine auxotrophs, a phenomenon that remains tobe understood but might be related to the previously describedpartial isoleucine and valine auxotrophy of cpx mutants (43).

Since a phenotype depends on the altered function of a geneproduct, we initiated a search for individual substrates of foldingfactors and proteases (Fig. 2 and 3). We concentrated on outermembrane proteins, as this class of cell envelope proteins has beenpreviously investigated (11, 48, 61). In agreement with publishedevidence, we detected a strong phenotype of the surA single mu-tation, i.e., decreased levels of LamB, OmpC, and OmpF (53, 59,71). In addition, our data reveal that a subpopulation of LamB

exists as unfolded monomers in degP dsbA, degP tsp, and degP yfgCdouble mutants. These unfolded monomers would normally en-ter the degradation pathway. However, in the protease mutantstrains the unfolded monomers are stabilized but remain assem-bly incompetent.

Furthermore, the dsbA null mutant expressed lower levels ofseveral outer membrane proteins containing Cys residues, includ-ing LptD, LamB, and FhuA. Surprisingly, however, OmpW wasalso affected, even though it does not contain Cys residues. It hasbeen reported previously that other Cys-less proteins such as theporin PhoE and the periplasmic protein MdoG are present atlower levels in dsbA mutants (26). Several explanations for thiseffect must be considered because these data do not allow us todistinguish between direct and indirect effects. A direct effectcould be caused by a chaperone activity of DsbA, which was shownto assist in the refolding of chemically denatured proteins lackingCys residues (73). Since the model of chaperone activity for DsbAis not widely accepted, alternative models suggest, for example,that defects in the cell envelope of dsbA strains prevent a set ofproteins from folding correctly in the periplasm (26, 70). An in-direct effect of the dsbA mutation on OmpW levels might be theinterference of LPS biogenesis that is caused by a reduction of Imp(LptD) levels. Even though the involvement of LPS in the biogen-esis of OmpW is unknown, this scenario seems not unlikely. Inany case, because OmpW levels are also reduced in degP tsp dou-ble-null strains, our data suggest that the small monomeric porinOmpW does not fold well in the periplasm and might thus repre-sent a suitable model for studying the assisted folding of outermembrane proteins.

It will be interesting to determine in future experiments the

FIG 4 Overview of synthetic phenotypes. Arrows indicate synthetic negativephenotypes of the double mutants (Tables 1 and 2). Colors represent growthconditions: black, rich medium; red, high temperature; blue, hyperosmolarityin combination with high temperature; green, SDS/EDTA; and orange, mem-brane defect.




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underlying molecular mechanisms of the detected lethal pheno-types of single mutants and the synthetic lethality of double mu-tants that were detected under specific growth conditions. Theseexperiments should be designed to clarify whether lethality resultsfrom the loss of function of specific substrates of protein qualitycontrol factors, from the accumulation and aggregation of mis-folded polypeptides or protein fragments, from alterations in lipidcomposition or destabilization of the murein layer, or from neg-ative effects on the protein translocation systems for periplasmicand outer membrane proteins or lipopolysaccharides.

ACKNOWLEDGMENTS

We are grateful to the National BioResource Project (NIG, Japan) for E.coli knockout strains, to Jon Beckwith, Volkmar Braun, Xaunxian Peng,Tom Silhavy, and Jan Tommassen for providing antisera, and to BarryBochner and Melisa Merdanovic for discussions.

M.E. was supported by the Deutsche Forschungsgemeinschaft and theFonds der Chemischen Industrie.

The funders had no role in the study design, data collection and anal-ysis, decision to publish, or preparation of the manuscript.

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