Vol. 175, No. 11JOURNAL OF BACrERIOLOGY, June 1993, p. 3327-33340021-9193/93/113327-08$02.00/0Copyright X 1993, American Society for Microbiology

Signal Sequence Processing Is Required for the Assembly ofLamB Trimers in the Outer Membrane of Eschenichia coli

JOHN H. CARLSON AND THOMAS J. SILHAVY*

Department of Molecular Biology, Lewis Thomas Laboratory, Princeton University,Princeton, New Jersey 08544-1014

Received 14 January 1993/Accepted 2 April 1993

Proteins destined for either the periplasm or the outer membrane of Escherichia coli are translocated fromthe cytoplasm by a common mechanism. It is generally assumed that outer membrane proteins, such as LamB(maltoporin or X receptor), which are rich in 13-structure, contain additional targeting information that directsproper membrane insertion. During transit to the outer membrane, these proteins may pass, in soluble form,through the periplasm or remain membrane associated and reach their final destination via sites of innermembrane-outer membrane contact (zones of adhesion). We report lamB mutations that slow signal sequence

cleavage, delay release of the protein from the inner membrane, and interfere with maltoporin biogenesis. Thisresult is most easily explained by proposing a soluble, periplasmic LamB assembly intermediate. Additionally,we found that such lamB mutations confer several novel phenotypes consistent with an abortive attempt by thecell to target these tethered LamB molecules. These phenotypes may allow isolation of mutants in which theprocess of outer membrane protein targeting is altered.

We wish to understand how proteins are targeted to theouter membrane of gram-negative bacteria. We have chosenEscherichia coli protein LamB (maltoporin) for our modelsystem. This protein is part of the maltose transport system(21). As a trimer, it functions as a specific channel toaccelerate the diffusion of maltose and maltodextrins acrossthe outer membrane permeability barrier. It also serves asthe receptor for bacteriophage X. By analogy with otherporin proteins, it is assumed that LamB exists as a 3-barrelstructure with 1-strands traversing the outer membrane (17).Little is known of the mechanism by which such proteins areinserted into the membrane. Isolation of mutants that arespecifically defective in outer membrane targeting shouldprovide a means to address this important question.Export of LamB and periplasmic maltose-binding protein

MalE, another component of the maltose transport system(21), has been analyzed extensively (3). These studies helpeddemonstrate that periplasmic and outer membrane proteinsare translocated from the cytoplasm by a common mecha-nism. Proteins destined for both locations are synthesizedinitially in precursor form with a typical signal sequence atthe amino terminus. The signal sequence directs the precur-sor to the cellular secretion machinery, which is composedprincipally of the sec gene products. During or shortly aftertranslocation, the signal sequence is removed by leaderpeptidase, producing the mature species (6). Thus, thetargeting pathways for periplasmic and outer membraneproteins diverge at a step after translocation.

It is generally accepted that the periplasm is the defaultpathway; proteins that contain no other targeting signals aresecreted into the periplasm, where they remain. Presumably,outer membrane proteins carry additional targeting informa-tion. However, attempts to locate this information by usingdeletion or gene fusion analyses have been largely unsuc-cessful (1, 17, 18); perhaps it is contained within a three-dimensional structure.Recent results from several laboratories suggest that outer

* Corresponding author.

membrane proteins are first secreted into the periplasm andthen inserted into the outer membrane by a second reaction(17, 18). Such insertion may or may not require additionalcellular factors. Although this hypothesis is attractive, theresults do not exclude an alternative proposal. Outer mem-brane proteins may always remain membrane associated,passing from the inner membrane to the outer membranethrough zones of adhesion.While it is clear that signal sequence cleavage is not

necessary for translocation from the cytoplasm, it is requiredfor release into the periplasm. Fikes and Bassford (10)constructed a malE mutant that prevents the processing ofMalE, resulting in tethering of the unprocessed molecule tothe cytoplasmic membrane. Despite this tethering, the pre-cursor form of MalE is functional. Apparently, the foldedmolecule retains sufficient mobility to interact with othercomponents of the transport system, although the amino-terminal end is anchored to the cytoplasmic membrane.The LamB and MalE signal sequences are similar, and we

reasoned that an analogous, processing-defective mutationwould likewise tether LamB to the cytoplasmic membrane.However, in this case, tethering might interfere with outermembrane targeting and, thus, LamB function. Resultspresented here show that this is the case. More importantly,we found that cells carrying the processing-defective lamBmutation exhibit several novel phenotypes which appear toreflect the abortive attempt by the cell to target the mutantLamB protein to the outer membrane. Such phenotypes mayprovide a means to identify mutants in which outer mem-brane protein targeting is altered.

MATERIALS AND METHODS

Strains and media. Strain construction was done as previ-ously described (14, 23). The strains and plasmids used inthis study are described in Table 1. Media used in this studywere prepared as previously described (23), except thatsugars in the liquid minimal media were at a final concentra-tion of 0.4%.

Site-directed mutagenesis. Mutagenesis of the lamB signal

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3328 CARLSON AND SILHAVY

TABLE 1. Strains and plasmids

Strain Genotype Source or reference

CC142 BAR1091 (ECB526 [MC4100 lac' lacIflF' lacIq TnS] malEA312) malE24-1 7ECB594 MC4100 malBA15/F+::Tn1O Spencer BensonJ134 HfrC XA reL41 spoTi T2' (ompF627fadL701) lacIq ApspA-C::kan 26JHC216 MC4100 malBAl lamB1869 This studyJHC222 MC4100 lamBA23D1869 rpoB argE(Am) supF/F'::TnJO This studyJHC224 MC4100 1amBA23D1869 This studyJHC247 JHC224 rpoB argE(Am) supF This studyJHC285 RAM1009 lamBA23D This studyJHC286 RAM1009 ApspA-C::kan This studyJHC287 JHC285 ApspA-C::kan This studyJHC296 RAM1009 malE24-1 This studyJHC308 RAM1009 lamBA78A23D Tjhis studyJHC510 MC4100 rpoB argE(Am) This studyJHC513 JHC51OsupF This studyJHC516 JHC510 supP This studyMC4100 F- araD139 A(argF-lac)U169 rpsL150 reLUI fibB5301 deoCI ptsF25 rbsR thi 23NT1001 MC4100 malBAl Nancy TrunNT1001 F+::TnlO NT1001 F+::TnlO This studyOC1500 MC4100 AlamB106 ompFA118-130 ompC198::TnlO 2RAM1009 MC4100 zjb::TnlO Rajeev MisraRAM1021 MC4100 1amB1869 Rajeev MisrapKBZ9000 pKBZ9000 19pDJA100 pKBZ9000 lamB24(Am) Dolores JacksonpJHC1 pKBZ9000 lamBA25P This studypJHC2 pKBZ9000 lamBA23D This studypJHC3 pJHC2 lamB5(Am) This studypJHC8 pJHC2 lamBA78 This study

sequence was done essentially as described by Rasmussen chromosome. Filamid lysates prepared on strains harboringand Silhavy (19). All of the plasmids used are derivatives of the mutant plasmid were used to transduce NT1001pKBZ9000 and carry the lamB-lacZ42-1 gene fusion. Table (F+::TnlO). NT1001 carries the malBWl deletion and is2 contains the name of the parent plasmid, the sequence of Mal-. Mal' derivatives can be isolated by demanding thethe oligonucleotide used to create the desired mutation, the homologous recombination event depicted in Fig. 1A. Thisresulting plasmid name, and the new lamB allele created. recombination event requires that any lamB signal sequenceThe general scheme utilized for all mutant construction mutation(s) on the plasmid be recombined into the chromo-

begins with a lamB-lacZ42-1-carrying plasmid containing an some, since the malBAl end point extends into the regionamber mutation located near the codon that is to be mutated. that encodes the mature portion of LamB. A Mal' pheno-Such strains form white (Lac-) colonies in the presence type requires a functional malK gene. LamB function is notof 5-bromo-4-chloro-3-indolyl-3-D-galactopyranoside (XG). required for growth on maltose but is required for growth onOligonucleotides were designed to create the desired muta- maltodextrins (the Dex phenotype). Therefore, mutationstion and simultaneously revert the amber codon to its that interfere with LamB function will not affect the selec-wild-type sequence. After mutagenesis, the putative mutant tion. P1 lysates were grown on the Mal' isolates and used toplasmids were transformed into ECB594. Plasmids contain- transduce NT1001 to Mal'. These strains, which carry noing the desired mutation were easily identified, because plasmid, were used for subsequent analyses.transformants form blue (Lac') colonies in the presence of Determination of Mal' recombination frequencies. StrainsXG. Introduction of an amber mutation was done in a similar were grown to saturation in LB (23) with ampicillin (125manner, except that white transformants were selected. pg/ml) at 37C. Cultures were centrifuged at 1,200 x g for 10Isolates containing the desired mutant plasmid typically min, the supernatant was removed, and cell pellets wereappeared at a frequency of 1 to 5%. The mutant plasmids suspended in an equal volume of M63. The suspended cellswere purified by preparing rvl filamid lysates on the selected were then diluted 10-1 and 10-2 in M63. A 100-,ul volume ofisolates and subsequent transduction into ECB594. each dilution was plated onto maltose minimal agar and

Introduction of lamB signal sequence mutations into the incubated at 37°C for 2 days. Mal' recombination frequen-

TABLE 2. Oligonucleotides used to construct mutant plasmids

Starting plasmid Oligonucleotide sequence (5' -3')a (newlamB allele)

pDJA100 CCG TGG AAA TCA ACA GGC ATT GCC TGA GC pJHCl (lamBA25P)pDJA100 GAA ATC AAC AGC CAT ATC CTG AGC AG pJHC2 (lamBA23D)pJHC2 GGA AGT TTG CGC TAA GTA ATC ATC pJHC3 (lamBS(Am)A23D)pJHC3 GCC CGC TGC GACAAGG AAG TTT GCG CAG AGT pJHC8 (lamBA78A23D)

a Bold letters indicate base changes from the wild-type sequence. The delta indicates the site of the A78 deletion.

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LamB TARGETING TO THE OUTER MEMBRANE 3329

A.

B.

'1m

PIXMI

lam1'

x

Ix

I laoZ

EEIk~a1 CHROWDSO]iEImalBAlI

TetR

lamB + ,j7DONOR

X X

I-fw<-x | RECIPIENTA28D 1869

FIG. 1. The recombination events used to introduce lamB signalsequence mutations into the chromosome. (A) Strategy used torecombine mutations from plasmids into the chromosome. Thesegment of plasmid DNA shown is the region containing E. coliDNA. 4, lamB-4acZ42-1 fusion joint; x in lamB, any desiredlamB signal sequence mutation. (B) Strategy used to recombinelamBA23D into the chromosome under noninducing conditions. TheP1 donor DNA was from strain RAM1009, and the recipient DNAwas from JHC224.

cies were determined by assuming a cell density of 2 x 109ml-1 in the undiluted sample.DNA sequence analysis. DNA sequence analysis was per-

formed with purified single-stranded plasmid DNA (19), orthe polymerase chain reaction was used to amplify chromo-somal DNA sequences (20). Polymerase chain reactionprimers (24 nucleotides) were designed to amplify a se-

quence extending from the translational start of malK to aposition 150 bp downstream of the coding region for theLamB signal sequence cleavage site. The sequencing primer(21 nucleotides) used hybridizes 150 bp upstream of thelamB translation start site.

Biochemical studies. Pulse-chase experiments, trimer anal-ysis, membrane fractionation by isopycnic sucrose densitygradient centrifugation, and immune precipitation were con-

ducted essentially as described by Misra et al. (15). Crudeouter membrane proteins were isolated by a variation of theprotocol previously described (16). Sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis and autoradiogra-phy were done as described by Stader et al. (24). Radioactivebands were quantitated with a densitometer (ScanMaker;MicroTek) and image analysis software (NIHImage), allthrough Macintosh computer programs.

Disk sensitivity analysis. Strains were inoculated into glu-cose minimal and maltose minimal media and grown tosaturation at 30°C. The cells were pelleted at 1,200 x g for 10min, the supernatant was removed, and the pellets were

suspended in an equal volume of M63. A 100-,u volume ofcells was mixed with 3 ml of F-Top agar and plated ontoglucose, glycerol, and maltose minimal agar. Antibiotic disks(Difco Dispens-o-disc System) were then placed on top ofthe solidified agar. For SDS disk sensitivity assays, 0.25-in.(1 in. = 2.54 cm) filter paper disks (Schleicher & Schuell)were placed on top of the agar and 10 pl of 10% SDS was

TABLE 3. Mal' recombination frequencies

Plasmid-encoded lamB Relevant chromosomal Mal+allele (plasmid name) allele(s) recombnation

lamB+ (pKBZ900) malBAl 10-4lamBA25P (pJHC1) malBAl 10-4lamB24(Am) (pDJA100) malBAl 10-4lamBA23D (pJHC2) malBAl <10-9lamBA78A23D (pJHC8) malBAl 10-4lamBA23D (pJHC2) malBAl lamB1869 10-4lamBA23D (pJHC2) malBAl lamB1869 supF 10-4

a Determination of Mal+ recombination frequencies is described in Mate-rials and Methods.

added to each disk. After incubation overnight at 30'C,diameters of zones of inhibition were measured.

Protein sequence analysis. Membrane proteins were dis-played on an SDS-polyacrylamide gel as described above.The proteins were transferred to Immobilon (Millipore) asdescribed by the manufacturer and stained with Coomassiebrilliant blue. The protein of interest was cut from themembrane, and the NH2-terminal amino acid sequence wasdetermined with an Applied Biosystems, Inc., 473A appara-tus.

RESULTS

Rationale and mutant design. The signal sequence insertsitself into the cytoplasmic membrane in an orientation thatpositions the amino-terminal end in the cytoplasm (12, 13).Consequently, as demonstrated by Fikes and Bassford (10)for MalE, mutations that prevent signal sequence cleavagetether the secreted protein to the cytoplasmic membrane. Byusing an analogous approach, we sought to tether LamB tothe cytoplasmic membrane, reasoning that such a mutationmay interfere with outer membrane targeting.The consensus site for the signal sequence processing by

leader peptidase is AXA (25), with cleavage occurring car-boxy terminal to the second alanine. In the case of MalE,there is a cryptic consensus site immediately preceding, andpartially overlapping, the normal cleavage site; the sequenceis ASALA (8). Fikes and Bassford (10) were able to simul-taneously inactivate both sites by changing the middle ala-nine codon to an aspartate codon, creating the allelemalE24-1. Like MalE, LamB has a similar cryptic cleavagesite; the sequence is AQAMA (5). In similar fashion, wechanged the middle alanine codon to an aspartate codon,which resulted in creation of the allele lamBA23D. Thismutation was constructed and verified by DNA sequenceanalysis as described in Materials and Methods. Biochemicalanalyses presented below demonstrated that this mutationinhibits LamB signal sequence processing as expected.LamBA23D is toxic. To recombine lamB signal sequence

mutations from plasmids into the chromosome, we used thegenetic scheme depicted in Fig. 1A (see Materials andMethods). With export-defective mutations or mutationsthat interfere with processing only slightly (lamBA25P), thisstrategy typically yielded Mal' recombinants at frequenciesof -10-4 (Table 3). However, when this approach was usedto introduce the lamBA23D allele, no Mal' recombinantswere observed (Table 3). This implies that the unprocessedmolecule is toxic to the cell. Since similar problems were notreported with the analogous malE24-1 allele (7), it appearsthat the toxicity relates to LamB.

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3330 CARLSON AND SILHAVY

LamiMall

lamB | lamB1869 IamBA23D186910' 30' 1 2' 3' 10 30" 1 2' 3' 4' 10" 30" 2' 3' 4'

wow_-"LamIl:l

FIG. 2. The lamBA23D mutation hinders signal sequence pro-cessing. The strains were pulsed for 20 s, and aliquots were taken atthe indicated post-chase time points. Samples were immune precip-itated and processed as described in Materials and Methods. Thestrains used were MC4100 (lamB+), RAM1021 (lamB1869), andJHC224 (1amBA23D1869).

Signal sequence mutations that internalize LamB alsoblock processing but cause no ill effects, suggesting that thetoxicity of the LamBA23D protein is manifested upon secre-tion. To test this hypothesis, a doubly mutant lamB signalsequence was constructed to prevent secretion of the mu-tant protein from the cytoplasm. The lamBA78 deletionremoves four amino acids in the hydrophobic core of thesignal sequence and blocks secretion at an early step (9).When Mal' recombinants were selected by using thelamBA78A23D double mutant plasmid, the recombinationfrequency returned to 10-4 (Table 3). We conclude from thisthat the toxicity of LamBA23D is evidenced during, orsubsequent to, translocation from the cytoplasm.The LamBA23D mutant protein may be toxic to the cell

because it is poorly translocated, hindering the vital processof protein secretion. Alternatively, the mutationally alteredsignal peptide may be a competitive inhibitor of leaderpeptidase, an essential protein (6). These possibilitiesseemed unlikely, since no such problems were noted withthe analogous MalE24-1 mutant protein (10). Rather, wesuspected that the harmful effects were the consequence oftethering LamB to the inner membrane. To address thisissue, we used malBAl mutant strain JHC216, which con-tains the amber mutation lamB1869. Strains containing thisnonsense mutation produce a truncated, inactive LamBmolecule of approximately 36 kDa that is efficiently translo-cated from the cytoplasm. The lamBA23D-containing plas-mid was introduced into an F+::TnlO derivative of thisstrain, and Mal' recombinants were demanded. As can beseen in Table 3, the recombination frequency returned to10-4. Similar results were obtained with nonsense mutationslocated at many different sites within lamB (data not shown).Thus, truncation prevents the toxicity associated with thelamBA23D mutation.The nontoxic nonsense fragment produced by strains

carrying the lamB1869 amber mutation can be easily de-tected by immunological methods. This allows direct bio-chemical testing of the effects of the 1amBA23D1869 muta-tion on processing and the secretion of other wild-typeproteins, such as MalE, with a standard pulse-chase assay.Results presented in Fig. 2 demonstrate that the lamBA23Dmutation inhibits LamB processing. In addition, this exper-iment shows that the secretion and processing of wild-typeMalE is unaffected. The simplest explanation for the resultspresented in this section is that the toxicity conferred by thelamBA23D mutation is manifested by tethered, full-lengthLamB on the periplasmic side of the cytoplasmic membrane.However, since the amber fragments are unstable, thepossibility remains that toxicity may be masked by degrada-tion.

Toxicity requires high-level synthesis of LamBA23D. Asnoted above, the toxicity associated with lamBA23D can be

A.

aati

B.

0 2 4 6 8 10 0 2 4 6 8 10

Time (hr:s TimesthrisFIG. 3. High-level synthesis of LamBA23D protein causes a

growth defect. Cells grown to saturation in glycerol minimal mediawere harvested and inoculated into glycerol (A) or maltose (B)minimal media to give a starting optical density at 600 nm (OD6.) of0.060 to 0.090. Cultures were then grown at 30°C. The strains usedwere RAM1009 (lamB+), JHC285 (lamBA23D), and JHC296(malE24-1).

relieved by a nonsense mutation. However, in no case werewe able to restore toxicity with nonsense suppressors.Several different combinations of nonsense mutations andsuppressors were tested. Even in situations in which thecorrect amino acid was inserted at the nonsense codon, no illeffects were observed. Recombination frequencies werenormal (data for lamB1869 are presented in Table 3), and allresulting strains grew normally under inducing conditions.Pulse-chase analysis showed that the suppressed strainsproduced full-length, unprocessed LamBA23D at levels thatapproached 50% of wild-type levels in certain cases (data notshown). We therefore conclude that the toxicity occurs onlywhen synthesis of LamBA23D occurs at high levels.The data presented in the previous paragraph suggest that

it is possible to introduce the lamBA23D mutation into thechromosome under noninducing conditions. The schemedevised for this construction is shown in Fig. 1B. Wereasoned that Tetr transductants, in which the desired re-combination event had occurred, could be identified byscreening for sensitivity to growth on maltose minimalmedium (Mal'). In no case did we observe transductantsincapable of growth on maltose. However, we did observetransductants that grew poorly on maltose at reasonablefrequencies (0.11%). These strains formed small colonies onmaltose-containing media but grew normally on all othermedia. Several such transductants were isolated, and thepresence of the lamBA23D mutation was verified by thestandard pulse-chase assay. One such mutant strain waschosen for further study, and its characterization is de-scribed below.Mals phenotype oflamBA23D mutant strains. To quantitate

the lamBA23D Mal' phenotype, growth assays were con-ducted at 30°C in glycerol (noninducing) and maltose (induc-ing) minimal media. The lamBA23D mutant strain grew aswell as its isogenic lamB+ and malE24-1 counterparts inglycerol minimal media (Fig. 3A). However, the lamBA23Dstrain exhibited a much longer lag period than either of thecontrol strains in maltose minimal media (Fig. 3B). The

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LamB TARGETING TO THE OUTER MEMBRANE 3331VOL. 175, 1993

TABLE 4. Disk sensitivity assays

Diam (mm) of zones of inhibition' measured on:

Allela Glucose minimal Glycerol Maltose minimalmedium minimal medium medium

lamB+ 0, 16, 0 0, 17, 0 0, 17, 0lamBA23D 0, 16, 0 0, 18, 0 11, 21, 0malE24-1 0, 16, 0 0, 18, 0 0, 17, 0lamBA78A23D 0, 16, 0 0, 18, 0 0, 17, 0ompFA118-130 14, 17, 17 18,,19, 19 17, 18,,18

a The representative strains used were RAM1009 (lamB+), JHC285(lamBA23D), JHC296 (maIE24-1), JHC308 (lamBA78423D), and OC1500(ompFA118-130).

b The values are diameters of zones of inhibition where no cell growthoccurred and are averages of three experiments conducted on different days.Diameters were rounded to the nearest whole number. The values in eachcolumn represent zones of inhibition created by SDS (10 pl of a 10%concentration), polymyxin B (300 U), and penicillin G (10 U), respectively.The values are from cells grown to saturation in glucose minimal media andused to inoculate each plate. The sensitivities observed after cells were grownto saturation in maltose minimal media were very similar. Experiments wereconducted at 30'C.

length of this lag period varied from experiment to experi-ment (from -2.5 to 8 h), at which point the cells began togrow at a constant rate. This rate never equaled the rateobserved with either the lamB+ or malE24-1 strain. Appar-ently, the mutant cells eventually adapted to the presence ofthe inducer. However, cycling of cells between inducing andnoninducing media revealed that this adaptation is epige-netic. Each time cells were returned to inducing media,adaptation was required (data not shown).The lamBA23D strain was capable of utilizing maltodex-

trins as a carbon source (Dex+), although the same inducersensitivity was observed. In addition, the lamBA23D muta-tion did not prevent infection by bacteriophage X, even onnoninducing media. This indicates that at least some LamBprotein reached the outer membrane and attained functionalform.

High-level synthesis ofLamBA23D perturbs the outer mem-brane. As noted above, the toxicity caused by high-levelsynthesis of the LamBA23D protein may be the result of theabortive attempt by the cell to target the mutant protein tothe outer membrane. Perhaps the mutant protein interfereswith the localization of other components to the outermembrane. Alternatively, it may be that the presence ofunprocessed LamB in the outer membrane is deleterious tothe cell. In either case, the integrity of the outer membranecould be altered. Since it is known that defects in the outermembrane can be evidenced by altered permeability, weexamined the sensitivity of the lamBA23D mutant strain tovarious antibiotics and detergents under inducing and non-inducing conditions. Selected results are shown in Table 4.

Results presented in Table 4 show that the strain contain-ing the lamBA23D mutant allele, but not an isogenicmalE24-1 strain, exhibited a dramatic increase in sensitivityto the detergent SDS, but only under inducing conditions.Again, this increased sensitivity requires secretion ofLamBA23D; signal sequence mutations relieve this pheno-type. The increased permeability observed was not general.We observed no increase in sensitivity to either hydrophilic(penicillin G) or hydrophobic (polymyxin B) antibiotics(Table 4) or to other detergents, such as deoxycholate (datanot shown). Increased sensitivity to SDS indicates an alteredouter membrane, and we conclude that LamBA23D, eitherdirectly or indirectly, disrupts the integrity of this membranebilayer.

lamB"3M 2' 5' 10' 'W

_- owd*

lamBA23O2' 5 10' 30'

tr4Lers

LamB.~~ ~ ~ ~ ~ ~ ~~~pLmLe"|1 h,M~~~~~~~~~~~~~MIFIG. 4. Trimer analysis of the lamBA23D mutant strain. Strains

were pulsed for 20 s, and aliquots were taken at the indicatedpost-chase time points. Samples were immune precipitated andprocessed as described in Materials and Methods. The strains usedwere RAM1009 (lamB+) and JHC285 (lamBA23D).

We considered the possibility that precursor LamBA23Dcan reach the outer membrane and form a larger LamB pore.This is plausible, since it is known that certain porin muta-tions, i.e., ompFA118-130, cause increased sensitivity toSDS (2). However, Table 4 shows that increased pore sizeresulted in increased sensitivity to penicillin G (as well asmany other compounds [2]). We did not observe a similarpattern of sensitivity with lamBA23D mutant strains. Ac-cordingly, we think this explanation unlikely.

Biochemical characterization of the lamBA23D mutantstrain. LamB trimer formation occurs in the outer membrane(15), and this provides a means to monitor the rate at whichthe protein reaches its final cellular location. Figure 4demonstrates that LamB trimers were detected as early as 2to 5 min postsynthesis, while comparable trimer amounts inthe lamBA23D background were not detected until the30-min time point. This demonstrated that the lamBA23Dmutation slows the rate of LamB trimer formation, whichcould be a consequence of impaired targeting of the proteinto the outer membrane.

Figure 4 also shows that the processing defect caused bythe lamBA23D mutation is leaky. Indeed, processing waslargely complete within 10 min. This result was verified byusing a standard pulse-chase assay (data not shown). Theprocessing defect caused by lamBA23D is much less severethan the corresponding defect caused by the analogousmalE24-1 mutation. With the malE mutation, we detected noprocessing, even after a 60-min chase, by which time pre-cursor degradation was apparent (data not shown). We donot understand the reason for this difference. However,amino acid sequence analysis did show that the LamBA23Dmolecule was processed correctly (data not shown).

Figure 4 shows that processing precedes trimer formation,which could indicate that processing is required for outermembrane targeting. However, this experiment does notclearly address the question of whether or not the unproc-essed LamBA23D molecule reaches the outer membrane.To determine the cellular location of the unprocessed

LamBA23D protein, mutant cells were labeled and fraction-ation experiments were performed following a 2-min chaseby using standard methods. As a control, we used isogeniclamB+ cells that had been treated in an identical fashion.The total amounts of LamB (precursor plus mature form)present in both mutant and wild-type cells were comparable,and the protein was found in the insoluble membrane frac-tion (data not shown). As summarized in Table 5, matureLamB in both strains was located primarily in the outermembrane, as expected. However, precursor LamB from

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3332 CARLSON AND SILHAVY

TABLE 5. Percentages of pLamB and LamB in cytoplasmic andouter membrane fractionsa

% in % in outerAllele Protein cytoplasmic membrane

membrane

lamB+ pLamB NDb NDLamB 19 81

lamBA23D pLamB 34.2 65.8LamB 19 81

a The values shown are percentages of the protein species found in therelevant membrane fractions. The strains were pulsed for 30 s and chased for2 min, at which point they were killed. Analysis was done as described inMaterials and Methods. The strains used were RAM1009 (lamB+) andJHC285 (lamBA23D).

b Precusor LamB protein was not detectable after 2 min of pulse-chase, asall of it is processed in a wild-type strain.

the lamBA23D mutant strain was found in both the cytoplas-mic and outer membranes.The fractionation result described above is difficult to

interpret. It could mean that at least some of the precursorform of LamBA23D reaches the outer membrane. Alterna-tively, the precursor molecule may be associated with boththe cytoplasmic and outer membranes simultaneously, or itmay be present in unusual membrane structures, such aszones of adhesion, in the cell. Cell breakage might thenpartition the precursor molecule into both membrane frac-tions randomly. We see no easy way to distinguish thesepossibilities but suggest that the precursor form ofLamBA23D must interact, in some way, with the outermembrane. In any event, the lamBA23D mutation clearlyslows the rate of trimer formation. Indeed, trimer formationappears to require processing.

Induction of LamBA23D induces the PspA stress shockresponse. The phenotypes conferred by the lamBA23D mu-tation could reflect an alteration in the protein compositionof the outer membrane. This possibility was tested byexamining outer membrane fractions of the mutant strainbefore and after induction. The fractionation protocol uti-lized, although somewhat crude, provides a simple andreproducible indication of outer membrane protein content.Results presented in Fig. 5 show that LamBA23D inductionhad no obvious effect on the steady-state levels of the majorouter membrane proteins. We conclude that LamBA23D hasno effect on the targeting of other outer membrane proteins.Note that the amount of processed LamB present in theouter membrane of the mutant strain at steady state wasconsiderable (30 to 50% of the wild-type level). This explainsthe Dex+ Xs phenotypes of the mutant strain.

Following growth in maltose minimal medium, a new,26-kDa protein appeared in the partially purified outer mem-brane fraction of the lamBA23D mutant strain (Fig. 5, lane4). This protein was not induced in cells carrying theanalogous malE24-1 mutation, nor was it induced in theompFA118-130 mutant, which also exhibited the SDSS phe-notype (data not shown). Moreover, it disappeared whenlamBA23D mutant cells, which had grown to saturationunder inducing conditions, were subcultured into glucoseminimal medium, grown to saturation, and treated as before(Fig. 5, lane 6). Thus, we conclude that the appearance of the26-kDa protein is not the result of a secondary mutation,which activates expression of the protein, but is rather theconsequence of cellular stress caused specifically by high-level synthesis of LamBA23D.To identify the 26-kDa protein, the NH2-terminal amino

1 2 3 4 5 6Cals $<Q>s SXS t;oe;y* fi 07t0<... S..... ov. .+.r*: CS, +E .,n..r ,_ X, H 0.0.V...; ,.. 0 i

., .. ..., .. $..

$0 0t701 0.<**;,t,>o<,t'0+a>M: ;ffftCr>00 ;JigSSSS

PspA'FIG. 5. LamBA23D synthesis induces the phage shock re-

sponse. Crude membrane protein fractions were isolated and ana-lyzed as described in Materials and Methods. Lanes: 1, 3, and 5,strain RAM1009 (lamB'); 2, 4, and 6, strain JHC285 (lamBA23D); 1and 2, cells grown to saturation in glucose minimal media; 3 and 4,cells grown to saturation in maltose minimal media; 5 and 6, cellsgrown to saturation in maltose minimal media (shown in lanes 3 and4) but subcultured into glucose minimal media and grown to satu-ration. All cell growth occurred at 30'C. The bands of interest arenoted at the sides. Lanes 1, 3, and 5 were overloaded to determinewhether PspA could be detected. Proteins were visualized bystaining with Coomassie blue.

acid sequence was determined. Results show that the first 21amino acids correspond to PspA, a stress response proteininduced by synthesis of filamentous bacteriophage proteinpiV and other cellular stresses, such as extreme heat andhigh ethanol concentrations (4). Apparently, all of thesecellular insults cause effects that are sensed by a commonmechanism. Previous studies have suggested that PspA is aperipheral inner membrane protein (4). We do not under-stand why it appears inlthese crude outer membrane prepa-rations. Perhaps this cross-contamination reflects the largeamount of PspA that is present in the cell.The appearance of PspA in induced lamBA23D mutant

cultures raised the possibility that this protein amelioratesthe deleterious effects of LamBA23D. The lag in growthobserved when the mutant was presented with maltose (Fig.3) might reflect the period required for PspA induction. Totest this hypothesis, we introduced a defined psp null mu-tation (ApspA-C::kan) into the lamBA23D mutant strainand checked each of the relevant phenotypes. No effectwas observed. The Psp response does not provide the cellwith an effective defense against high-level synthesis ofLamBA23D.

DISCUSSION

Although signal sequence cleavage is not required fortranslocation from the cytoplasm, it is necessary for releaseof secreted proteins from the inner membrane. With MalE, aperiplasmic protein, mutations that block processing tetherthe precursor molecule to the cytoplasmic membrane (7, 10).This poses little problem for the cell; indeed, the anchoredmolecule is functional. We would expect similar results forother periplasmic proteins. For outer membrane proteins,signal sequence processing and release from the inner mem-brane are likely required for functional assembly. The pro-cessing defect caused by lamBA23D hinders trimer forma-tion; in fact, it may block it completely (see below).Moreover, this defect has dire consequences for the cell.Mutant cells are inducer sensitive, and high-level synthesisof the mutant protein alters the integrity of the outer mem-

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LamB TARGETING TO THE OUTER MEMBRANE 3333

brane and causes induction of the phage shock stress re-sponse. We predict similar results for other major outermembrane proteins, and this might explain problems previ-ously encountered with certain ompA (11) and ompF (27)mutant constructs.The inducer-sensitive phenotype of lamBA23D strains is

not severe. Mutant cells eventually adapt and grow in thepresence of maltose. However, even after adaptation, cellsshow signs of distress; growth rates are reduced, and thephage shock stress response is induced to a high level. ThelamBA23D mutation is quite leaky; processing occurs but isdelayed, and a significant amount of mature LamB eventu-ally reaches the outer membrane. This leakiness may permitcell survival in the presence of an inducer. Indeed, wesuspect that a total processing block would be lethal. Testingof this prediction is difficult, owing to the presence of acryptic processing site. Deletions could result in a totalblock, but they may also interfere with signal sequencefunction.The requirement for adaptation in the presence of an

inducer probably explains why the lamBA23D mutationcould not be recombined directly into the chromosome onmaltose minimal agar. We do not understand the mechanismof adaptation. Clearly, the proteins of the phage shock stressresponse are not involved. It is possible that adaptationrequires production of a periplasmic protease that degradesa fraction of the mutant protein or catalyzes cleavage at thenormal processing site. However, we have been unable toobtain evidence supporting such a mechanism.We assume that the lamBA23D mutation, like its counter-

part malE24-1, tethers the mutant protein to the innermembrane. In the case of the nonsense fragment producedby the lamBA23D1869 double mutant, this seems to be so; itis localized largely in the cytoplasmic membrane. However,fractionation analysis with the full-length LamBA23D mu-tant protein is difficult to interpret; two-thirds of the unproc-essed molecule appears to fractionate with the outer mem-brane. Whether or not this reflects the actual cellulardistribution of the mutant protein cannot be determined. Themutant protein may be associated with both membranessimultaneously. Likewise, we do not know whether theunprocessed LamBA23D mutant protein is assembled intotrimers. Because processing precedes trimer formation, wethink this unlikely. If some assembly does occur, the struc-ture must be unstable. At steady state, the only speciesdetectable in the outer membrane is mature, wild-typeLamB.To account for our results, we propose the simple model

presented in Fig. 6. According to this hypothesis, mutationsthat block processing tether the secreted protein to the innermembrane. This causes no problems with periplasmic pro-teins, such as MalE, which have no additional targetinginformation. Outer membrane proteins, such as LamB, carryadditional targeting information, and outer membrane inser-tion commences. Because the protein remains anchored inthe inner membrane, the targeting process cannot be com-pleted, and the protein remains stuck at some intermediatestage with the outer and inner membranes joined together inabnormal fashion. Sufficient numbers of abnormal structuresare toxic to the cell and are lethal if not resolved by signalsequence cleavage. The model accounts for the followingfindings. (i) The inducer-sensitive phenotype is not severe,and cells survive because the processing defect is leaky. (ii)Trimer formation cannot occur until the abnormal structureis resolved, i.e., the signal sequence is processed. (iii) Thisabnormal structure perturbs the integrity of the outer mem-

-tl IOM

t CMFIG. 6. Proposed model explaining the phenotypes exhibited by

the lamBA23D mutant strain. The protein shown represents ageneric outer membrane (OM) protein. The black box depicted inthe cytoplasmic membrane (CM) represents the uncleaved signalsequence, and the curvy line in the outer membrane depicts themature portion of the outer membrane protein.

brane in a manner that increases sensitivity to SDS. (iv) Theambiguous results of fractionation experiments reflect thefact that cellular breakage disrupts the abnormal structureand randomly partitions the mutant protein to both mem-brane fractions. More is found in the outer membranefraction because the interaction between the mutant proteinand the outer membrane is stronger. (v) The phage shockstress response is induced because the inner and outermembranes are brought into close proximity. Alternatively,it may be induced as a consequence of some alteration of theadhesion zones observed in wild-type cells.As noted near the beginning of this report, two models for

outer membrane protein biogenesis have received seriousconsideration. The first proposes that these proteins are firstsecreted into the periplasm and then inserted into the outermembrane in a second reaction. The second model invokeszones of adhesion as sites for selective diffusion of theseproteins from the inner membrane to the outer membrane.Our data do not clearly distinguish between these possibili-ties, but we favor the former. (i) It is difficult to imagine howouter membrane proteins that are hydrophilic and rich in vstructure could be inserted into the inner membrane. (ii) Ablock in signal sequence cleavage would not necessarilyimpede passage of a protein through an adhesion zone;tethering is certain to cause problems if release into theperiplasm is required. (iii) The number of LamB moleculesfar exceeds the number of adhesion zones in a normal cell. Ifunprocessed LamB interfered with the function of the zones,we would expect low-level synthesis to be toxic. (iv) Mostimportant, it has been shown biochemically that denaturedouter membrane porin proteins can be inserted into outermembranes in vitro (22).

It is possible, of course, that outer membrane insertionoccurs spontaneously in vivo. However, given that natureappears to leave little to chance, we favor the idea thatcellular components participate in the targeting reaction.The novel phenotypes exhibited by lamBA23D mutantstrains (Mals SDS5) may provide a means to select formutants in which targeting is altered. For example, muta-tions that slow this reaction may relieve these phenotypes byallowing increased time for signal sequence cleavage.An alternative model to explain the toxicity conferred by

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3334 CARLSON AND SILHAVY

lamBA23D proposes that the unprocessed molecule eventu-ally reaches the outer membrane and disrupts its integritydirectly. While this possibility cannot be excluded, we thinkit unlikely. Mutations in ompF, which cause sensitivity toSDS and other agents (2), are not toxic to the cell, nor dothey induce the phage shock stress response. Even if thismodel is correct, the phenotypes exhibited by the lamBA23Dmutant should still allow selection for mutants defective inouter membrane targeting. Mutations that prevent the pro-tein from reaching this destination should relieve the defects,and it would not be difficult to exclude uninteresting nullmutations or mutations that impede translocation of themutant protein from the cytoplasm.

ACKNOWLEDGMENTS

We thank laboratory members, especially Dolores Jackson andRajeev Misra, for critical comments. Marjorie Russel providedstrain J134, and the Phil Bassford laboratory provided strain CC142.Mark Flocco performed amino acid sequence analysis and synthe-sized the oligonucleotides used in this study.

This work was supported by a grant from the U.S. Public HealthService to T.J.S.

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