7
Vol. 175, No. 1 JOURNAL OF BACTERIOLOGY, Jan. 1993, p. 222-228 0021-9193/93/010222-07$02.00/0 Copyright © 1993, American Society for Microbiology Role of the Carboxyl-Terminal Domain of TolA in Protein Import and Integrity of the Outer Membrane SHARYN K. LEVENGOOD-FREYERMUTH, EVA MARIE CLICK, AND ROBERT E. WEBSTER* Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710 Received 6 August 1992/Accepted 21 October 1992 The TolA protein is involved in maintaining the integrity of the outer membrane of Escherichia coli, as mutations in toL4 cause the bacteria to become hypersensitive to detergents and certain antibiotics and to leak periplasmic proteins into the medium. This protein also is required for the group A colicins to exert their effects and for many of the filamentous single-stranded bacteriophage to infect the bacterial cell. ToIA is a three-domain protein, with the amino-terminal domain anchoring it to the inner membrane. The helical second domain is proposed to span the periplasmic space to allow the carboxyl-terminal third domain to interact with the outer membrane. A plasmid that allowed the synthesis and transport of the carboxyl-terminal third domain into the periplasmic space was constructed. The presence of an excess of this domain in the periplasm of a wild-type cell resulted in an increased sensitivity to deoxycholate, the release of periplasmic alkaline phosphatase and RNase into the medium, and an increased tolerance to colicins El, E2, E3, and A. There was no effect on the cells' response to colicin D, which depends on TonB instead of ToIA for its action. The presence of the free carboxyl-terminal domain of ToIA in the periplasm in a toUt null mutation did not restore the wild-type phenotype, suggesting that this domain must be part of the intact TolA molecule to perform its function. Our results are consistent with a model in which the carboxyl-terminal domain of ToIA interacts with components in the periplasm or on the inner surface of the outer membrane to function in maintaining the integrity of this membrane. The cell envelope of the gram-negative bacterium Esch- erichia coli is a complex organelle. It is composed of an outer membrane and an inner or cytoplasmic membrane as well as the aqueous periplasmic space between them con- taining the murein sacculus (6, 22, 24). These structures create a formidable barrier to the uptake of macromolecules from the extracellular medium. Only small molecules (<600 Da) are able to pass freely through the outer membrane into the periplasm, and most of these molecules then require some type of active transport system to cross the inner membrane. Many of the proteins found in the bacterial envelope are involved in such import mechanisms. Addition- ally, there are proteins which function in the synthesis and maintenance of the envelope structure itself. A variety of bacteriophage and bacterial toxins have taken advantage of some of these resident envelope proteins to enter the bacteria. For example, TonB and its auxiliary proteins ExbB and ExbD serve to promote vitamin B12 and siderophore-mediated iron transport into the bacterium (5, 26). However, these same proteins are used by bacterio- phage T1 and )80 for infection of the bacterium and by the group B colicins (including B, D, Ia, Ib, M, and V) for translocation to their sites of action (7, 26). Similarly, most or all of the TolQRAB proteins are required for infection by the filamentous bacteriophage (fl, M13, fd, and Ike) and for sensitivity to the group A colicins (including El, E2, E3, A, K, L, and N) (8, 21, 23, 31, 32). Although no transport function has been assigned to these Tol proteins, bacteria which contain mutations in the tol genes are hypersensitive to detergents and to certain antibiotics, and they release periplasmic proteins into the medium (14, 32). The pheno- types of these mutants suggest that the Tol proteins are involved in maintaining the integrity of the outer membrane. * Corresponding author. tol mutants also exhibit a tolerant phenotype, such that colicin or phage binding to specific outer membrane recep- tors is normal but subsequent translocation of these mole- cules to their respective sites of action is inhibited. In bacteria containing mutations in tolQ, toiR, or toiB, this tolerant phenotype can be overcome by raising the concen- tration of the colicin approximately 103- to 104-fold (5, 32). However, tol mutants can withstand concentrations of colicins 106-fold higher than that needed to kill tolA + strains, indicating that the TolA protein may play a major and unique role in the uptake of the bacteriophage and colicins. To begin to elucidate the function of the Tol proteins, we have focused on characterization of the ToLA protein. An analysis of the structure and cellular location of the 421-amino-acid TolA protein showed that it is composed of three domains (16). The amino-terminal 47 amino acids, domain I, include a 20-residue hydrophobic membrane- spanning region which serves to anchor the protein in the inner membrane. The remaining carboxyl-terminal 348 amino acids reside in the periplasm. This region is composed of two domains: the carboxyl-terminal 120 residues, desig- nated domain III, and the central 254 amino acids, desig- nated domain II. This latter region appears to be a continu- ous helix acting as a rigid tether connecting domains I and III. The structure of TolA suggests that domain III may interact with periplasmic or outer membrane proteins as part of TolA function. Interaction of domain III with the inner surface of the outer membrane and/or peptidoglycan layer would result in a ToLA bridge between these structures and the inner membrane. Such a bridge structure might aid in transport of components to the outer membrane. In addition, it would allow domain III to interact with a specific colicin after it had bound to its receptor. This hypothesis would be consistent with the results of Benedetti et al. (3) showing that colicins A and El are capable of binding TolA in vitro. 222 on December 29, 2020 by guest http://jb.asm.org/ Downloaded from

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Vol. 175, No. 1JOURNAL OF BACTERIOLOGY, Jan. 1993, p. 222-2280021-9193/93/010222-07$02.00/0Copyright © 1993, American Society for Microbiology

Role of the Carboxyl-Terminal Domain of TolA in ProteinImport and Integrity of the Outer Membrane

SHARYN K. LEVENGOOD-FREYERMUTH, EVA MARIE CLICK, AND ROBERT E. WEBSTER*

Department ofBiochemistry, Duke University Medical Center, Durham, North Carolina 27710

Received 6 August 1992/Accepted 21 October 1992

The TolA protein is involved in maintaining the integrity of the outer membrane of Escherichia coli, as

mutations in toL4 cause the bacteria to become hypersensitive to detergents and certain antibiotics and to leakperiplasmic proteins into the medium. This protein also is required for the group A colicins to exert their effectsand for many of the filamentous single-stranded bacteriophage to infect the bacterial cell. ToIA is a

three-domain protein, with the amino-terminal domain anchoring it to the inner membrane. The helical seconddomain is proposed to span the periplasmic space to allow the carboxyl-terminal third domain to interact withthe outer membrane. A plasmid that allowed the synthesis and transport of the carboxyl-terminal third domaininto the periplasmic space was constructed. The presence of an excess of this domain in the periplasm of a

wild-type cell resulted in an increased sensitivity to deoxycholate, the release of periplasmic alkalinephosphatase and RNase into the medium, and an increased tolerance to colicins El, E2, E3, and A. There wasno effect on the cells' response to colicin D, which depends on TonB instead ofToIA for its action. The presence

of the free carboxyl-terminal domain of ToIA in the periplasm in a toUt null mutation did not restore thewild-type phenotype, suggesting that this domain must be part of the intact TolA molecule to perform itsfunction. Our results are consistent with a model in which the carboxyl-terminal domain of ToIA interacts withcomponents in the periplasm or on the inner surface of the outer membrane to function in maintaining theintegrity of this membrane.

The cell envelope of the gram-negative bacterium Esch-erichia coli is a complex organelle. It is composed of anouter membrane and an inner or cytoplasmic membrane aswell as the aqueous periplasmic space between them con-taining the murein sacculus (6, 22, 24). These structurescreate a formidable barrier to the uptake of macromoleculesfrom the extracellular medium. Only small molecules (<600Da) are able to pass freely through the outer membrane intothe periplasm, and most of these molecules then requiresome type of active transport system to cross the innermembrane. Many of the proteins found in the bacterialenvelope are involved in such import mechanisms. Addition-ally, there are proteins which function in the synthesis andmaintenance of the envelope structure itself.A variety of bacteriophage and bacterial toxins have taken

advantage of some of these resident envelope proteins toenter the bacteria. For example, TonB and its auxiliaryproteins ExbB and ExbD serve to promote vitamin B12 andsiderophore-mediated iron transport into the bacterium (5,26). However, these same proteins are used by bacterio-phage T1 and )80 for infection of the bacterium and by thegroup B colicins (including B, D, Ia, Ib, M, and V) fortranslocation to their sites of action (7, 26). Similarly, mostor all of the TolQRAB proteins are required for infection bythe filamentous bacteriophage (fl, M13, fd, and Ike) and forsensitivity to the group A colicins (including El, E2, E3, A,K, L, and N) (8, 21, 23, 31, 32). Although no transportfunction has been assigned to these Tol proteins, bacteriawhich contain mutations in the tol genes are hypersensitiveto detergents and to certain antibiotics, and they releaseperiplasmic proteins into the medium (14, 32). The pheno-types of these mutants suggest that the Tol proteins areinvolved in maintaining the integrity of the outer membrane.

* Corresponding author.

tol mutants also exhibit a tolerant phenotype, such thatcolicin or phage binding to specific outer membrane recep-tors is normal but subsequent translocation of these mole-cules to their respective sites of action is inhibited. Inbacteria containing mutations in tolQ, toiR, or toiB, thistolerant phenotype can be overcome by raising the concen-tration of the colicin approximately 103- to 104-fold (5, 32).However, tol mutants can withstand concentrations ofcolicins 106-fold higher than that needed to kill tolA + strains,indicating that the TolA protein may play a major and uniquerole in the uptake of the bacteriophage and colicins. To beginto elucidate the function of the Tol proteins, we havefocused on characterization of the ToLA protein.An analysis of the structure and cellular location of the

421-amino-acid TolA protein showed that it is composed ofthree domains (16). The amino-terminal 47 amino acids,domain I, include a 20-residue hydrophobic membrane-spanning region which serves to anchor the protein in theinner membrane. The remaining carboxyl-terminal 348amino acids reside in the periplasm. This region is composedof two domains: the carboxyl-terminal 120 residues, desig-nated domain III, and the central 254 amino acids, desig-nated domain II. This latter region appears to be a continu-ous helix acting as a rigid tether connecting domains I andIII.The structure of TolA suggests that domain III may

interact with periplasmic or outer membrane proteins as partof TolA function. Interaction of domain III with the innersurface of the outer membrane and/or peptidoglycan layerwould result in a ToLA bridge between these structures andthe inner membrane. Such a bridge structure might aid intransport of components to the outer membrane. In addition,it would allow domain III to interact with a specific colicinafter it had bound to its receptor. This hypothesis would beconsistent with the results of Benedetti et al. (3) showing thatcolicins A and El are capable of binding TolA in vitro.

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PROTEIN IMPORT AND tolA 223

In this report, we show that TolA function requires theinteraction of domain III with periplasmic components byexamining the in vivo effects of removing or overproducingTolA domain III (TolA-III) in the bacterial cell. Neithersoluble domain III in the periplasm nor TolA lacking domainIII was able to substitute for intact TolA. However, thepresence of excess soluble domain III in the periplasm madewild-type bacteria leaky for periplasmic proteins, hypersen-sitive to deoxycholate, and more tolerant to the group Acolicins. The data suggest that domain III of intact TolA isinvolved in functional interactions with envelope constitu-ents. These phenotypes are consistent with the hypothesisthat domain III of TolA, when connected to the innermembrane via domain II, interacts with components on theinner surface of the outer membrane to exert its function.

MATERIALS AND METHODS

Bacterial strains and plasmids. E. coli K17 (C600 Strr lac),K38 (HfrC Strr), K91 (a X- derivative of K38), K964 (K91containing pColE2), and K828 (K91 containing pColE3)were obtained from Marjorie Russel and Karen Jakes (TheRockefeller University). RK4441 containing pColD was pro-vided by R. Kadner (University of Virginia), DM1187 con-taining pMY440, which overproduces colicin El, was ob-tained from W. Cramer (Purdue University), and C600containing pColA9 (18) was provided by R. Lloubes (CentreNational de la Recherche Scientifique, Marseilles, France).K17(DE3) is K17 lysogenized with X DE3 carrying theinducible gene for T7 RNA polymerase, and K17Al(DE3)contains a mini-TnlO insertion near nucleotide 200 of thetolA gene (16). Plasmids pET3c and pLysS (27) as well as XDE3 (29) were obtained from F. W. Studier (BrookhavenNational Laboratory). Lysozyme produced from pLysSreduces the basal level activity of T7 RNA polymerase.Plasmid pDNC186 was obtained from P. Bassford (TheUniversity of North Carolina, Chapel Hill). This plasmidcontains rbsB, the structural gene for the ribose-bindingprotein (RBP), under the control of the lacUV5 promoter(10, 19).

Plasmid pSKL10 containing tolA under the control of theT7 promoter, plasmid pSKL17 encoding domains II and IIIof TolA (Fig. 1A), and plasmid pSKL19 encoding domain IIIof TolA are described by Levengood et al. (16). PlasmidpSKL21, encoding domain III of ToIA preceded by a signalsequence (Fig. 1B), was constructed by first digestingpSKL10 with NdeI and NotI and isolating the 4.9-kb NdeI-NotI fragment containing the 3' end of tolA plus vectorsequences encoding ampicillin resistance and the 410 pro-moter. This fragment was ligated together with an NdeI-NotI106-bp fragment encoding the RBP signal sequence. This106-bp fragment was obtained by amplifying the appropriateregion of pDNC186 with AmpliTaq DNA polymerase (Per-kin-Elmer Cetus) according to the manufacturer's protocol.Two primers, complementary to opposite strands of theDNA sequence and flanking the region to be amplified, weresynthesized with additional nucleotides on their 5' ends sothat the resulting amplified DNA contained a 5' NdeI siteand a 3' NotI site. The pSKL21 plasmid resulting from thisligation encodes a protein containing the signal sequence forRBP and five amino acids of the mature RBP protein,including the cleavage site for leader peptidase, joined todomain III of TolA (Fig. 1B). Plasmid pSKL22, encodingdomains I and II of TolA (TolA-I,II) (Fig. 1C), was preparedby cleaving pSKL10 with NotI, filling in the overhangingends with the Klenow fragment, and then religating the

A. ToIA protein

domain(47 aa)

domain 11(254 aa)

domain III(120 aa)

LIM O

Im ~~~~~~~~~Om

B. pSKL21 (RBP-ToIA-I)

RBP signalsea

(25 aa)RBP start

ToIA domain 1II

(131 aa)

ToIA domain III

(5 aa) (131 aa)

pre-RBP-ToIA-III(16,329 Da)

RBP-ToIA-III(13,856 Da)

C. pSKL22 (ToIA-l,II)

domain i

(47 aa)

domain 11

(248 aa)

+1AGA-Gi-JCAGAUGA UAUU

FIG. 1. Schematic representations of TolA, RBP-ToIA-III, andTolA-III. (A) The TolA protein is presented as three domains asdescribed by Levengood et al. (16). The amino terminus is to theleft. _, hydrophobic membrane-spanning region; , predictedhelical region. The location of TolA relative to the inner (IM) andouter membrane (OM) is shown. aa, amino acids. (B) The product ofplasmid pSKL21 is the fusion of the signal sequence of RBP and thecarboxyl-terminal 131 amino acids (aa) of TolA (upper diagram). Itis designated pre-RBP-ToIA-III. The lower diagram depicts theprotein following secretion into the periplasm. It is designatedRBP-TolA-III and consists of the amino-terminal five amino acids ofthe mature RBP fused to the carboxyl-terminal portion of TolA. (C)The product of pSKL22, designated ToIA-III, contains domain Iand 248 amino acids (aa) of domain II of TolA. The right-handportion shows the RNA sequence near the terminating codon forthis protein. The codon for the last amino acid is underlined. Theproposed +1 ribosome frameshift is shown by the line above thesequence. The box is around a potential Shine-Dalgarno sequence.

DNA. This resulted in a four-nucleotide insertion creatingthree in-frame stop codons near the end of domain II.Media and chemicals. Bacteria were routinely grown in TY

(tryptone-yeast) medium or LB (Luria broth) medium con-taining glucose or maltose when required as described bySun and Webster (30). When necessary, antibiotics wereused in the following concentrations: ampicillin, 60 ;xg/ml;chloramphenicol, 15 jig/ml; and tetracycline, 20 jig/ml. IPTG(isopropyl-,-D-thiogalactopyranoside) and XP (5-bromo-4-chloro-3-indolyl phosphate) were obtained from ResearchOrganics, hen egg lysozyme and Torula yeast RNA type VIwere purchased from Sigma Chemical Co., and 125I-labeledprotein A was purchased from Dupont. Restriction endonu-cleases and DNA-modifying enzymes were purchased fromBoehringer Mannheim Biochemicals and Bethesda ResearchLaboratories. Colicins E2, E3, and A were prepared asculture supernatants of mitomycin-induced bacteria contain-ing the requisite Col plasmids (4, 18). Colicin El wasprepared from DM1187 carrying pMY440 by carboxymeth-yl-Bio-Gel A column chromatography (28). The final con-

--MMP

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224 LEVENGOOD-FREYERMUTH ET AL.

centration of the purified El was 8 mg/ml in 0.1 M NaPO4(pH 7.0). Colicin D was produced in RK4441 by inductionwith mitomycin (300 ,ug/ml) for 3 h. The bacteria wereharvested by centrifugation, washed in 0.05 M NaPO4 (pH7.0), and broken by sonication. The resulting lysate wassubjected to centrifugation at 100,000 x g for 3 h, and thesupernatant containing colicin D was stored at -20'C.Growth curves and Western immunoblot analysis. K17

(DE3) or K17Al(DE3) containing either pSKL21 or pSKL22together with pLysS was grown in LB medium containingthe appropriate antibiotics to a density of approximately 2 x108 cells per ml. The cultures were not induced or wereinduced with various concentrations of IPTG. The opticaldensity at 600 nm of the samples was recorded every 15 minfollowing induction. At 1 and 2 h following induction, 1-mlsamples were removed and the cells were pelleted andresuspended in 200 p.l of sample buffer (2% sodium dodecylsulfate [SDS], 1% P-mercaptoethanol, 5% glycerol, 0.004%bromophenol blue, 0.125 M Tris-HCl [pH 6.8]). The proteinsin each sample were separated by SDS-polyacrylamide gelelectrophoresis (PAGE), transferred to nitrocellulose, andsubjected to Western blot analysis using anti-TolA-II,IIIantibody and 125I-labeled protein A as a probe (16). Toquantitate the amount of RBP-TolA-III present, differentconcentrations of a purified protein containing the carboxyl-terminal 117 residues of TolA fused to 14 amino acids ofvector sequence (16) were run on the same gel and subjectedto the same Western blot analysis. The amount of radioac-tivity was determined for each band and plotted versus theconcentration. Comparison of the radioactivity in the RBP-TolA-III band from each sample with this standard curve

allowed an approximation of the RBP-TolA-III present inthe bacteria.

Cellular fractionations. Cells were grown to a density ofapproximately 2 x 108/ml and then induced with IPTG forthe times indicated. The bacteria in 1-ml samples were

collected, resuspended in 200 ,ul of sample buffer, and heatedat 90°C for 3 min. This fraction is referred to as whole cells.The remaining cells were then harvested in a Sorvall SS34rotor for 10 min at 5,000 rpm. The cell pellet was resus-

pended in 10 mM Tris-HCl (pH 8)-20% (wt/wt) sucrose. Themixture was stirred on ice as lysozyme was added to a finalconcentration of 100 pLg/ml. After 2 min of incubation on ice,an equal volume of 10 mM Tris-HCl (pH 8)-i mM EDTAwas slowly added to the stirring mixture of cells to allowspheroplast formation. The spheroplasts were then collectedby centrifugation in an SS34 rotor for 10 min at 10,000 rpm.Five milliliters of the supernatant was removed and precip-itated with trichloroacetic acid to a final concentration of5%. The precipitate was collected, washed with cold ace-

tone, and resuspended in 0.5 ml of sample buffer as de-scribed above. This fraction contains the components of theperiplasm. The spheroplast pellet was resuspended in 10 mMTris-HCl (pH 8)-0.5 mM EDTA and put through a 27-gaugeneedle several times to enhance lysis. Following centrifuga-tion of the sample for 10 min at 5,000 rpm to remove anyunbroken spheroplasts, the supernatant was centrifuged in a

TY65 rotor at 45,000 rpm for 3 h. Five milliliters of thesupernatant was collected, and the proteins were precipi-tated by addition of trichloroacetic acid to 5% and resus-

pended in 0.5 ml of sample buffer. This fraction contains thecytoplasmic proteins. The pellet from the high-speed spinwas directly resuspended in 1.0 ml of sample buffer. Thisfraction is designated membranes. All fractions were sub-jected to SDS-PAGE and Western blotting as describedabove.

Trypsin was used to analyze the topography of the TolAconstructs associated with the membrane (16). Bacteriacontaining pSKL22 and pLysS were grown to a concentra-tion of 2 x 108 cells per ml in 50 ml of LB and induced for 15min with 0.4 mM IPTG. The bacteria were harvested andincubated 10 min at 00C in 5 ml of 8.55% (wtlvol) sucrose-1mM EDTA-0.03 M Tris-HCl (pH 8.0). The cells werecollected by centrifugation and resuspended in 5 ml of 8.55%(wt/vol) sucrose-0.01 M Tris-HCl (pH 8.0), and then 1-mlaliquots were treated with trypsin (25 puglml) or buffer for 1h at 14WC. Trypsin inhibitor was added to 25 ,ug/ml; then thecells were collected and resuspended in 0.2 ml of samplebuffer. The proteins were separated by SDS-PAGE andanalyzed by the Western blot technique as described above.

Colicin sensitivity and outer membrane leakage. Bacteriacontaining the appropriate plasmids were grown to 1.5 x 108cells per ml and not induced or induced with 0.04 mM IPTGfor 1 h. To measure the sensitivity to colicins, 0.2-mlsamples of the culture were added to 3 ml ofTY soft agar andplated on freshly made TY plates containing the appropriateantibiotics. In addition to the antibiotics, the soft agar andplates used for the induced bacteria contained 0.04 mMIPTG. The soft agar was allowed to harden, and 2 pl of eachcolicin dilution was spotted onto the plate. The extent ofcolicin sensitivity was determined for each dilution by thesize and clarity of the spot following incubation at 37°C for 6to 8 h.The permeability of the outer membrane was measured by

determining the cells' relative susceptibility to deoxycholateand by detecting the release of periplasmic alkaline phos-phatase or RNase I into the medium. Bacteria were plated onTY medium containing 0.2% (wt/vol) deoxycholate, a con-centration at which the growth of wild-type E. coli isunaffected. Release of alkaline phosphatase was detected byplating bacteria on low-phosphate plates containing XP andmeasuring the size of the blue halo around each colony.Leakage of RNase I was determined by growing the bacteriaon TY plates containing 1.5% (wtlvol) RNA as described byLazzaroni and Portalier (15).

RESULTS

Synthesis of periplasmic domain Ill of TolA. Previousanalysis of TolA showed that it is an inner membrane proteincomposed of three structural domains (Fig. 1A) (16). Theposition of domain III at the end of the long helical domainII suggested that its normal function might be to interact withcomponents available in the periplasm. If this hypothesis iscorrect, then an excess of a soluble form of TolA domain IIIin the periplasm should compete with ToLA for recognition ofthese components and produce phenotypes similar to thoseof toLA mutant bacteria. To test this assumption, plasmidpSKL21 was engineered to encode the carboxyl-terminal 131amino acids of TolA joined to the signal sequence and leaderpeptidase cleavage site of RBP (Fig. 1B). Synthesis of thisprotein should lead to the secretion into the periplasm of a

soluble processed protein consisting of five amino acids ofRBP followed by the 131 carboxyl-terminal amino acids ofTolA (RBP-TolA-III). Expression of this gene from pSKL21under various conditions of induction leads to the productionof two proteins which can be detected by Western blotanalysis with anti-TolA-II,III antibody and which migrate atpositions expected for pre-RPB-TolA-III and RPB-TolA-III(Fig. 2). The amount of each protein in these bands was

determined by comparison with known amounts of a proteincontaining the carboxyl-terminal 117 residues of TolA fused

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PROTEIN IMPORT AND tolA 225

Ihour 2 hourNP 0.0 .04 .08 0.0 .04 .08

pSKL211 2 3 4 1 2 3 4

pDNC1861 9 A2 A N P Ind.

-ToI A66-

45--o

21- ,

14- ;1i

FIG. 2. Western blot of K17(DE3) cells containing pSKL21 andpLysS induced with various concentrations of IPTG. Bacteria weregrown to 2 x 108 cells per ml and induced with the amount(millimolar) of IPTG indicated above each lane. Sample NP did notcontain any pSKL21. Samples were taken at 1 and 2 h and subjectedto SDS-PAGE on a 10 to 16% gradient gel. The resulting gels weresubjected to Western blot analysis using anti-TolA-IIIII antibody asdescribed in Materials and Methods. This antibody is more reactiveto intact TolA than to proteins containing only TolA-III. Sizes areindicated in kilodaltons.

to 14 amino acids of vector sequence (16) subjected to thesame Western blot analysis. The results indicate that in theabsence of IPTG, approximately 10,000 molecules of thesmaller protein are produced per cell. This is presumably themature RBP-TolA-III protein. Very little if any of the largerprotein, presumed to be the uncleaved pre-RBP-TolA-III, ispresent under these conditions. Induction with 0.04 mMIPTG for 1 h increases the amount of RBP-TolA-III to40,000 molecules per cell and produces an additional 23,000molecules of the larger pre-RBP-TolA-III. Growth in liquidculture or on plates is unaffected by the presence of pSKL21and 0.04 mM IPTG. Higher concentrations of IPTG result ina slowing of the growth of the bacteria (data not shown)along with an accumulation of pre-RBP-TolA-III.The identities of the precursor and mature forms of

TolA-III encoded by pSKL21 were confirmed by cell frac-tionation (Fig. 3). The smaller protein was present in theperiplasm as expected for RBP-TolA-III, while the larger

W P C M

68kDao-", <TolA

31 kDa,

_pre-RBP-ToIA-I1114kDa,' RBP-ToIA-III

FIG. 3. Cellular fractionation of K17(DE3) cells containingpSKL21 and pLysS. Bacteria were grown to 2 x 108 cells per ml,incubated with 0.04 mM IPTG for 1 h, and fractionated intoperiplasm, membrane, and cytoplasm as described in Materials andMethods. The proteins in each fraction were subjected to SDS-PAGE and Western blot analysis using anti-TolA-II,III antisera asdescribed in the legend to Fig. 2. Wild-type TolA (44.2 kDa) ispresent in the membrane fraction. W refers to whole cell sampletaken after the bacteria were harvested and resuspended in sucrosesolution. The periplasmic (P), membrane (M), and cytoplasmicfractions (C) were isolated from the whole cell fraction.

ToIA-,_MBP-

RBP-EX

.a.

A B C DFIG. 4. Effect of pre-RBP-TolA-III on transport of RBP and

MBP. K17(DE3)/pLysS cells containing pSKL21 were grown to 2 x108/ml and incubated with 0.04 mM IPTG; samples were taken at 1and 2 h for Western blot analysis with anti-TolA-II,III (A) anti-MBP(B), and anti-RBP (C). Lanes: 1, bacteria without pSKL21; 2 and 3,pSKL21-containing bacteria induced for 1 and 2 h, respectively; 4,bacteria treated with 0.2 mM sodium azide to enhance production ofthe precursors (25). Positions of TolA, mature MBP, and matureRBP are shown. (D) Western blot analysis using anti-RBP ofK17(DE3)/pLysS without (NP) or with pDNC186 induced for 1 hwith 2 mM IPTG (Ind.). A SDS-10 to 16% gradient polyacrylamidegel was used; arrows indicate positions of standards with molecularsizes of 66, 45, 31, 21.5, and 14.4 kDa (from top to bottom).

protein was present in the cytoplasm as expected for theprecursor. The accumulation of the pre-RBP-TolA-III in thecytoplasm suggests that this is a result of rapid folding of thechimeric molecule into a form that is export incompetent.These molecules might impair the transport of otherperiplasmic proteins by the normal secretory pathway, per-haps by sequestering chaperone proteins such as SecB.However, there is no accumulation of precursors to maltose-binding protein (MBP) or RBP under conditions in whichpre-RBP-ToLA-III accumulates (Fig. 4). Thus, the bacteriacan tolerate the presence of the large amount of the precur-sor and processed TolA-III proteins resulting from inductionby 0.04 mM IPTG without any effect on either bacterialgrowth or the normal translocation of proteins into or acrossthe membrane.

Synthesis of membrane associated Tol-I,II. If domain III ofTolA is required for normal TolA function, then the TolA-III fragment missing this domain should not be able toreplace TolA in the bacteria. To test this assumption,pSKL22 was engineered to prematurely terminate synthesisof TolA producing truncated TolA-I,II (Fig. 1C). Synthesisof this protein in the presence of increasing amounts of IPTGresulted in increasing amounts of TolA-I,II (Fig. 5, lanes 3 to5). Cell growth appeared normal in the presence of 0.04 mMIPTG, and after 1 h there was about three to four times moreTolA-I,II than TolA found in wild-type bacteria. The TolA-III fragment exhibited the same sensitivity to trypsin inEDTA-treated bacteria as did intact TolA (Fig. 6; comparelanes 4, 8, and 12). Therefore, TolA-I,II is inserted into theinner membrane with the same topology as is TolA.

In addition to ToLA-I,II, there is a small amount of intactTolA which is synthesized from pSKL22 (Fig. 5, lane 5). Theratio of intact TolA to TolA-I,II is approximately 0.05 underthe conditions of induction shown. It is most likely that thesynthesis of TolA is the result of a + 1 ribosome frameshift-ing event at the position shown in Fig. 1C. Although thenucleotide sequence in the region of this presumptive frame-shift differs from that found in other sites known to besubject to frameshifting, the region is flanked by a 5'

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226 LEVENGOOD-FREYERMUTH ET AL.

68kDa,.

_0w ** 4TolA

43 kDa,

29kDai1 2 3

TolA- 1,11

4 5FIG. 5. IPTG-induced expression of ToIA-III from pSKL22.

Cell cultures were grown to 2 x 108 cells per ml, and then IPTG was

added as indicated below. After 1 h of induction, cells were

collected and lysed in sample buffer. The protein from an equalnumber of cells in each sample was subjected to SDS-PAGEfollowed by Western blot analysis using anti-ToIA-II,III as de-scribed in Materials and Methods. The radioactivity in each bandwas quantitated in an LKB Clini-gamma counter. Lanes: 1, toLA4+[K17(DE3)/pLysS] uninduced; 2, tof [K17Al(DE3)/pLysS] unin-duced; 3, toIA/pSKL22, uninduced; 4, toIA/pSKL22 plus 0.04 mMIPTG; 5, toIA/pSKL22 plus 0.08 mM IPTG.

Shine-Dalgarno sequence and a 3' UGA, both of which havebeen shown to act as stimulators for the ribosome shift (1).

Effect of the TolA fragments on membrane integrity andcolicin uptake. The presence of approximately 10,000 mole-cules of soluble periplasmic TolA domain III (RBP-TolA-III) or about 1,200 molecules of membrane-associated TolA-III had no effect on the membrane integrity of tolA nullbacteria, since large periplasmic proteins still leak into themedium and the bacteria are hypersensitive to deoxycholate(Table 1). Thus, neither fragment will substitute for intactTolA in maintaining the integrity of the membrane. How-ever, the presence of 10,000 molecules RBP-TolA-III in theperiplasm of a tolA + bacteria results in leakage of RNase andalkaline phosphatase into the medium, the phenotype of tolAbacteria (Table 1). Under these conditions, there is little ifany pre-RBP-TolA-III present in the bacteria (Fig. 2). Thischange to the mutant phenotype was enhanced in the pres-ence of 0.04 mM IPTG, when the ratio of periplasmicRBP-TolA-III to intact TolA was increased from 10- to

+ _+ + -_-_+ + -_-_+

68kDaC

43kDa,

29kDa,

1 2 3 4 5 6 7 8 9 10 11 12

FIG. 6. Membrane localization of TolA-I,II. K17(DE3) cellscontaining pLysS and either pSKL10, pSKL22, or pSKL17 were

grown to 2 x 108/ml and induced for 15 min with 0.4 mM IPTG. Thebacteria were treated with EDTA and trypsin as described inMaterials and Methods. Lanes: 1 to 4, K17(DE3) with pSKL10encoding TolA; 5 to 8, K17(DE3) with pSKL22 encoding TolA-III;9 to 12, K17(DE3) with pSKL17 encoding TolA-II,III. ToIA-II,III iscytoplasmic since it is missing the signal sequence in domain I (16)and thus is unaffected by trypsin. Lanes 1, 5, and 9 contain wholecells; all remaining lanes contain EDTA-shocked cells. Lanes 2, 6,and 10 represent 0-min time points after EDTA treatment; samplesin lanes 3, 4, 7, 8, 11, and 12 were taken after 1 h of incubation in theabsence (-) or presence (+) of trypsin.

TABLE 1. Effects of ToIA fragments on membrane integrity

Bacterial RNase I PhoA Sensitivity togenotype Plasmid Fragmenta leakage" leakagec deoxycholatedtoL4 + No No No

pSKL21 RBP-TolA-III Yese Yes YesepSKL22 TolA-III No No NopSKL10 TolA No NDf No

toLA Yes Yes YespSKL21 RBP-To1A-III Yes Yes YespSKL22 TolA-I,II Yes Yes YespSKL10 TolA No No No

a Bacteria were grown in the absence of IPTG and contained approximately10,000 molecules of periplasmic RBP-TolA-III and approximately 1,200molecules of TolA-I,II per cell as determined by Western blot analysis (seeMaterials and Methods). The same results were obtained in the presence of0.04 mM IPTG except as noted below (footnote e)."Colonies were grown on TY-antibiotic plates containing 1.5% yeast RNA,

and then the plate was layered with 5 ml of cold 10% trichloroacetic acid.RNase I leakage was detected as a clear halo around a colony.

c Colonies were grown on low-phosphate-antibiotic plates containing theenzyme substrate XP. Alkaline phosphatase (PhoA) leakage was detected asa blue halo.

d Growth was measured on TY-antibiotic plates containing 0.2% (wt/vol)deoxycholate. Yes, little or no growth (sensitive); No, normal growth (resis-tant).

e Phenotype enhanced by the presence of 0.04 mM IPTG in plates.f ND, not determined.

40-fold. These effects on the membrane integrity appearspecific to the production of soluble TolA-III. IPTG-inducedoverexpression of RBP from plasmid pDNC186 producesalmost 20% pre-RBP (Fig. 4D) yet does not result in leakageof RNase or hypersensitivity to deoxycholate when assayedin the same manner (data not shown). These results stronglysuggest that the soluble periplasmic domain III is able tocompete with the domain III of intact TolA for interactionswith specific components in the periplasm that are requiredfor the normal function of TolA.Such a competition also is reflected in an increased

tolerance to the effects of the group A colicins (Table 2). Thedegree of tolerance observed is directly proportional to theratio of soluble domain III to intact TolA as would beexpected if these molecules were competing for interactionwith the same component of the bacteria. In the case ofcolicins El and A, periplasmic RBP-TolA-III might beinteracting with the colicin itself, as indicated by the resultsof Bene-dtti et al. (3). The presence of RBP-TolA-III has noeffect on the sensitivity of the bacteria to colicin D, amember of the group B colicins that do not need the Tolsystem to exert their effects.The synthesis of TolA-I,II from pSKL22 in tolA bacteria is

not able to correct the leaky phenotype, showing that it wasnot able to substitute effectively for intact TolA function(Table 1). This is the case even when the cells contain athree- to fourfold excess of TolA-I,II (data not shown).However, these bacteria demonstrated various degrees ofincreased sensitivity to different group A colicins (Table 2).For example, in the presence of 0.04 mM IPTG, thesebacteria were almost as sensitive to El as were wild-typebacteria while retaining some tolerance to colicins E2 andE3. This reversal of the tolerant phenotype probably is notdue to the presence of the TolA-I,II fragment but rather is aresponse to the small amount of intact TolA which issynthesized from pSKL22 under these conditions (Fig. 5). Itis obvious that very little ToIA is required for colicin El toexert its effect, while much more is required for the action ofcolicins E2, E3, and A. Since both El and A exert their

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PROTEIN IMPORT AND tolA 227

TABLE 2. Effects of TolA fragments on colicin uptakea

Bacterial IPTG Molecules of Molecules of TolA Relative concn of colicind:genotype Plasmid (mM) TolA/celib fragment/cell' El E2 E3 A

toL4 + 800 1 1 1 1pSKL21 800 10,000 3 3 3 3pSKL21 0.04 800 40,000 10 6 6 10

tolA 0 > 107 > 103 > 103 > 104pSKL22 <50 1,200 6 103 103 103pSKL22 0.04 <200 3,000 1 102 102 27

a K17(DE3)/pLysS containing the indicated plasmid was grown to a concentration of 1.5 x 108 cells per ml. IPTG was added as indicated, and growth continuedfor 1 h. Aliquots (0.2 ml) were plated in soft agar, and 2 Ill of various dilutions of each colicin was spotted onto the plates as described in Materials and Methods.

b From Levengood et al. (16).Estimated by Western blot analysis as described in Materials and Methods.

d Expressed as the fold increase in concentration of colicin needed to produce the same clear spot size as in wild-type K17(DE3)/pLysS. A concentration of>101 indicates that the bacteria are tolerant to the highest concentration of colicin available in that preparation. For colicin D, a group B colicin which uses theton, not the tol, system, the relative value was 1 in all cases tested.

effects by acting as ionophores, their different requirementsfor TolA may reflect differences in their interactions with theTol system.

DISCUSSION

Previous experiments have shown that TolA is anchoredto the inner or cytoplasmic membrane~by its amino-terminaldomain I (16). The remaining portion of ToIA, comprisingdomains II and III, is contained within the periplasmicspace. Considering the extended helical nature of domain II,we suggested that it might act as a tether allowing domain IIIto interact with components present in the periplasm. Thedata presented in this report are consistent with such ahypothesis. When present in a soluble form in the periplasm,domain III can interfere with TolA function. This interfer-ence can be detected as an increased permeability of theouter membrane to large molecules and increased toleranceof the bacteria to group A colicins. The presence of solubleperiplasmic TolA domain III appears to disrupt only specificTolA functions. It has no effect on the normal secretion ofthe proteins across the cytoplasmic membrane or the abilityof colicin D, a group B colicin, to kill the bacteria. Inaddition, a similar overproduction of RBP does not lead to aleakage of periplasmic proteins into the medium.The TolA molecule must be intact for its function in

maintaining outer membrane integrity. It cannot be replacedeither by periplasmic domain III (RBP-TolA-III) or by themembrane-associated truncated TolA containing domains Iand II (TolA-I,II). The experiments with pSKL22 in the tolAstrain suggest that at least 200 molecules are required tomaintain membrane integrity. Fewer are required for theaction of colicins A and El, probably reflecting the fact thatso few molecules of these ionophores are needed to kill thebacteria. It is not known with what bacterial componentsdomain III interacts functionally. The long helical region ofdomain II would allow domain III to interact with moleculespresent on the inner surface of the outer membrane, creatinga connection between these membranes. However, thereprobably exists enough flexibility in the helix and the poly-glycine region between the domain I inner membrane anchorand the helical domain II tether to allow interaction ofdomain III with the peptidoglycan layer, soluble periplasmicproteins, and perhaps even molecules on the periplasmicface of the inner membrane. There also may be structuralconstraints to this flexibility if TolA is part of an oligomericstructure with itself and the TolQ, TolR, and TolB proteins.

The role that TolA plays in the import of the group Acolicins is not clear. From the data presented in this report,we propose that domain III of TolA may interact with thecolicin-receptor complex in the outer membrane as part ofthe translocation process. Presumably this involves a directinteraction between domain III and the colicin moleculeitself in the case of colicins A and El. Benedetti et al. (3)showed that colicins El and A were able to interact directlywith TolA in vitro and that this interaction was dependent onthe presence of an intact domain III. Colicin A was able tobind to a degradation product of TolA which was notrecognized by colicin El, suggesting a difference in thebinding interactions of these colicins with TolA domain III.

Since TolA is strictly required for the action of all group Acolicins, TolA must be able to interact with a number ofdifferent outer membrane receptor-colicin complexes. Oneof the difficulties in formulating any model of ToIA domainIII interaction with a colicin-receptor complex is our lack ofknowledge of the nature of these receptor-ligand complexes.Colicins E, A, and K and the filamentous phage vary in theirouter membrane receptor requirements as well as in theirrequirements for ancillary proteins necessary for their trans-location into or across the bacterial membranes (32). Forexample, in addition to the BtuB receptor protein, colicin Elrequires the outer membrane TolC protein and colicin Arequires the presence of OmpF. Both of these proteins are inthe outer membrane, but their exact role in any outermembrane receptor complex is not clear. A logical hypoth-esis might be that the interaction of colicins A and E withtheir respective outer membrane proteins results in specificdelivery of these colicins to domain III of TolA. The colicinsthen must interact with the other Tol proteins and perhapsdomain II of TolA to be translocated into or across thebacterial inner membrane.What is the role of TolA and the other products of the

tolQRAB gene cluster in the normal metabolism of E. coli? Itis possible that these proteins are involved in the uptake ofspecific molecules, similar to the role of the proteins in theTonB system. TonB is the major component of a high-affinity uptake system for iron siderophore complexes andvitamin B12 (5, 11, 26). It is also utilized by the group Bcolicins for their transport into the cell. TonB is anchored inthe inner membrane and is proposed to span the periplasmicspace as does TolA. However, in contrast to TonB, TolAand the other Tol proteins have not been implicated in theuptake of any essential nutrients under laboratory condi-tions. Since mutations in toLA affect outer membrane integ-

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228 LEVENGOOD-FREYERMUTH ET AL.

rity, whereas tonB mutations show no such effect, wepropose that the Tol proteins function in the transport ofnewly synthesized components to the outer membrane,helping to maintain its integrity. ToLA could play a structuralrole in this process by bringing together the inner and outermembranes to provide a direct link or bridge between thetwo membranes.TolA appears capable of interacting with outer membrane

components which in themselves are capable of multipleinteractions. For example, BtuB when serving as receptorfor the E colicins interacts with TolA and when serving asreceptor for vitamin B12 interacts with TonB. Therefore, theinteractions of TolA with specific outer membrane proteinssuch as BtuB to form a bridge between the two membranesmay be of a transient nature. Rapid fixing of bacteria mightimmobilize these structures, allowing visualization of theseinner and outer membrane connections by electron micros-copy. Such connectors or adhesion zones have been shownto exist by such fixation methods (2). Other types of fixation,such as cryofixation (12), might not stabilize any transientbridges formed between the membranes by such a TolA-directed noncovalent protein interaction. More biochemicaland genetic evidence about the location of the Tol proteinsand their interactions with other bacterial proteins is neededbefore their precise role in maintaining the integrity of theouter membrane can be determined.

ACKNOWLEDGMENTS

We thank Mary Jo Outlaw for help in processing the manuscriptand Gerda Vergara for assistance with the figures.

This work was supported by Public Health Service grantGM18305 from the National Institute of General Medical Sciences.

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