JOURNAL OF BAGrERIOLOGY, Sept. 1992, p. 5661-5668 Vol. 174, No. 170021-9193/92/175661-08$02.00/0Copyright C 1992, American Society for Microbiology

Enhanced Export of ,3-Galactosidase Fusion Proteinsin prlF Mutants Is Lon DependentWILLIAM B. SNYDER AND THOMAS J. SILHAVY*

Department ofMolecular Biology, Princeton University, Princeton, New Jersey 08544

Received 9 April 1992/Accepted 27 June 1992

We have used fusions of the outer membrane protein LamB to 13-galactosidase (encoded by lacZ) to study theprotein export process. This LamB-LacZ hybrid protein blocks export when synthesized at high levels, asevidenced by inducer (maltose) sensitivity, a phenomenon termed LacZ hybrid jamming. The priFI mutationrelieves LacZ hybrid jamming and allows localization of the fusion protein to a noncytoplasmic compartment.priFl and similar alleles are gain-of-function mutations. Null mutations in this gene confer no obviousphenotypes. Extragenic suppressors of a gain-of-function priF allele have been isolated in order to understandhow this gene product affects the export process. The suppressors are all Ion null mutations, and they areepistatic to all priF phenotypes tested. Lon protease activity has been measured in prlFI cells and shown to beincreased. However, the synthesis of Lon is not increased in a priFI background, suggesting a previouslyunidentified mechanism of Lon activation. Further analysis reveals that priFI activates degradation ofcytoplasmically localized precursors in a Lon protease-dependent manner. It is proposed that accumulation ofprecursors during conditions of hybrid protein jamming titrates an essential export component(s), possibly achaperone. Increased Lon-dependent precursor degradation would free this component, thus allowingincreased protein export under jamming conditions.

The protein export process in the bacterium Eschenichiacoli has been genetically characterized with the aid of genefusions. Specifically, hybrid proteins that contain amino-terminal fragments of an exported protein fused to 3-galac-tosidase have been created. The fusion protein used in thiswork contains the signal sequence and first 181 amino acidsof mature LamB fused to 3-galactosidase (Hyb 42-1) (27).This hybrid can be recognized by the protein export machin-ery as a substrate for the export process. Recognition ismediated by the amino-terminal signal sequence, a targetingsignal that has been functionally conserved between prokar-yotes and eukaryotes (25). Our laboratory studies export ofan outer membrane protein encoded by the lamB gene.LamB trimers form a porin in the outer membrane thatserves as the bacteriophage X receptor and is essential forthe uptake and subsequent utilization of maltodextrins, butnot maltose, by the cell. Work in other laboratories has madeuse of the malE gene, which encodes the periplasmic mal-tose-binding protein and is required for the uptake of bothmaltose and maltodextrins. These genes are part of the malBlocus, which encodes all components of the maltose trans-port system (reviewed in reference 26).Two novel phenotypes conferred by the lamB-lacZ or

malE-lacZ fusions have been exploited to genetically char-acterize the protein export process. First, localization of thehybrid proteins to a noncytoplasmic compartment leads todecreased ,-galactosidase activity that is probably due to theinability of the enzyme to assume an active conformation(22). This phenotype has been used successfully to identifygenes encoding components of the protein export machin-ery, the sec genes, through the isolation of conditional lethalmutants which exhibit increased P-galactosidase activity(reviewed in reference 5). Second, high-level synthesis of thehybrid protein results in lethality due to the incompatiblenature of P-galactosidase with the export process, a condi-

* Corresponding author.

tion known as hybrid jamming or maltose sensitivity (5). TheP-galactosidase portion of the hybrid, being derived from acytoplasmic protein, contains sequences or structures whichare incompatible with the export reaction (5, 15). Mutationsthat alter the hybrid protein signal sequence can result in aninability of the export machinery to recognize the hybrid,thus rendering the cells resistant to maltose (5). In suchstrains, P-galactosidase activity increases as a result oflocalization of the hybrid in the cytoplasm (5).The prlFl mutation was identified by its ability to render

fusion-containing strains maltose resistant (13). This extra-genic suppressor of hybrid protein jamming, unlike a signalsequence mutation, results in decreased P-galactosidaseactivity (13). This phenotype is due to increased export ofthe fusion protein from the cytoplasm; no alteration of thesteady-state levels of hybrid has been observed (13).

Additional alleles of prlF have been isolated by theirability to suppress the temperature sensitivity of a straincontaining a degP (htrA) null mutation (2). The degP (htrA)gene encodes a periplasmic protease that is essential for cellviability at temperatures above 39°C (16, 17, 32, 33). Sup-pressors of these temperature-sensitive mutants that restoregrowth at 42°C were identified. The complementing gene wasisolated and, through sequence analysis, shown to be anallele of prlF. These alleles of prlF were called sohAl andsohA2 (suppressor of htrA). With respect to hybrid proteinexport, the sohAl, sohA2, and prlFl mutants are phenotyp-ically identical (see below). In addition, all of these allelesconfer a cold-sensitive growth defect. The mutations elicit again of function with respect to protein export and coldsensitivity, since a mutant containing a null allele of the gene(prlF::kan) has neither phenotype (12).We have taken advantage of the cold sensitivity of sohA2

mutants to isolate cold-resistant extragenic suppressors.One class of cold-resistant suppressors that restores hybridprotein jamming and returns P-galactosidase activity towild-type levels has been characterized. Identification of thesuppressors and their effects on protein export have been

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5662 SNYDER AND SILHAVY

examined. The suppressor analysis suggests a possible rolefor PrlF in cellular physiology.

MATERIALS AND METHODS

Bacterial strains, phages, and plasmids. The strains of E.coli K-12 and the plasmid used in this study are described inTable 1.Media and chemicals. All media have been previously

described (26). Minimal medium was supplemented with0.2% sugar when required except for glycerol, which wasadded at 0.4%. Amino acids were supplemented at a finalconcentration of 0.03% when required for growth. To selectfor tetracycline sensitivity, we used the fusaric acid selectionmethod described previously (7). We obtained [35S]methio-nine from NEN Research Products, Du Pont Co., Boston,Mass., and Kodak XAR film from Eastman Kodak, Roch-ester, N.Y. Formalin-fixed Staphylococcus aureus (Immu-no-Precipitin) was purchased from Bethesda Research Lab-oratories, Gaithersburg, Md. 5-Bromo-4-choro-3-indolyl-3-D-galactoside (X-Gal) was obtained from Bachem, Torrance,Calif. Methyl methanesulfonate was purchased from East-man Kodak. o-Nitrophenyl-13-D-galactoside (ONPG) camefrom Sigma Chemical Co., St. Louis, Mo.

Genetic techniques. Standard techniques for P1 transduc-tion, bacterial conjugation, and bacteriophage lysate prepa-ration have been described elsewhere (20, 26).

,-Galactosidase assay. For the cpsB-lacZ assay, cells weregrown at 371C in liquid Luria broth to mid-log phase. Theassays were performed as described previously (29).

Maltose sensitivity assays. Sensitivity was measuredagainst 10 ,ul of 2% maltose on a filter paper disk atop a lawnof the test strain as described previously (6). Plates wereincubated at 37°C overnight.

Lysogenization assays. Test strains were grown overnightat 37°C in Luria broth supplemented with 0.2% maltose.Cells were pelleted and resuspended in 0.5 volume of 10 mMMgSO4; 100 ,ul of cells (-10" cells_per ml) was added to 100,ul of bacteriophage X (3.6 x 10 PFU/ml, as titered onWBS115) to give a multiplicity of infection of 3.6 x 10-4ensuring that only X was limiting for the assay. The cell andX mixture was incubated 20 min at 23°C and shifted to 37°Cby the addition of 800 pI of prewarmed Luria broth. Cellsand X were incubated at 37°C for 20 mmn before serial dilutionand plating onto LB plates spread with a high-titer lysate ofX cI and X cIh80 to select growth of lysogens. All dilutionmedia and plates were prewarmed to 37°C, and the experi-ment was completed at 37°C so that the cells would neverundergo any additional temperature shifts. The experimentswere performed in triplicate for each overnight culture of thegiven strains and were repeated from three overnight cul-tures grown on separate days. The percentage of lysogenformation is given as number of lysogens per input phage x100. The mean and standard deviation were calculated fromthese data.

Pulse-labeling and immunoprecipitation. Overnight cul-tures of test strains were grown at 37°C in liquid minimalmedia. For the analysis of precursor degradation, cells weregrown in glycerol minimal medium supplemented with Luriabroth to 0.5%. Cells were subcultured 1:50 into the samemedium and grown to an A600 of 0.2 to 0.3. Maltose wasadded to a final concentration of 0.8%, and cells were grownfor an additional hour prior to pulse-labeling. For the deter-mination of Lon synthesis, test strains were grown in glu-cose minimal medium overnight, subcultured into the samemedium, and grown to anA6. of -0.5. All cells were labeled

for 30 s with 10 ,uCi of [35S]methionine (> 1,000 Ci/mmol) perml of cells. Cells were chased with an equal volume ofprewarmed medium identical to the labeling medium supple-mented with 0.8% methionine. One milliliter of cells wascollected at various times postchase into 50 pul of 100%(wtlvol) trichloroacetic acid. Following >30 min of incuba-tion at 0WC, samples were pelleted in a microcentrifuge andwashed with 1 ml of cold (-20°C) acetone. The preparationof samples for immunoprecipitation and electrophoresis hasbeen described elsewhere (30). The anti-Lon antibodies werea gift from Susan Gottesman, National Institutes of Health,Bethesda, Md.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

(SDS-PAGE) and autoradiography. The techniques havebeen described previously (30).

Densitometry analysis. The technique has been describedelsewhere (30). To compensate for loading differences, theareas of all mature MalE peaks were normalized to the samevalue, and the remaining peaks in that lane were normalizedby the same factor.Immunoblot analysis. Cells were grown identically to cells

being prepared for pulse-labeling. One milliliter of cells waspelleted, resuspended in a volume of lx loading buffer (26)calculated by the formula volume (pJ) = A60J3.6, and boiledfor 5 min. Then 10 pul of each sample was subjected toSDS-PAGE and electroeluted to a nitrocellulose membraneas described previously (11), with the following modifica-tions. Transfer buffer 2 (25 mM Tris, 192 mM glycine, 20%methanol) was used to electroelute the proteins to nitrocel-lulose membranes (Schleicher & Schuell, Keene, N.H.),which were then blocked in MTS buffer (0.9% NaCl, 0.01 MTris-HCl [pH 7.5], 3% powdered milk) for 30 min. Anti-LamB and anti-MalE antibodies were diluted 1:1,000 in MTSand incubated with the nitrocellulose membranes for at least1 h with gentle shaking. Membranes were washed threetimes for 5 min each time with wash buffer (0.2% Tween,0.9% NaCl, 0.01 M Tris-HCl [pH 7.5]). Nitrocellulose wasreblocked with MTS prior to incubation for 1 h with horse-radish peroxidase-conjugated donkey anti-rabbit antibody(Amersham, Arlington Heights, Ill.) diluted 1:5,000 in MTS.Membranes were washed as before, and antibody detectionwas performed with the ECL detection reagents (Amer-sham) as described by the supplier.

RESULTS

Despite extensive characterization in two different labora-tories, including DNA sequence analysis, no obvious func-tion for PrlF has been revealed. To address this importantquestion, we wished to use suppressor analysis. Previousattempts to identify suppressor mutations by searching formutants of prlFl lantB-lacZ fusion strains that exhibitedincreased LacZ activity were unsuccessful. Most, if not all,of the mutants identified carried either an additional prlFmutation that simply inactivated theprlFl gene product or asignal sequence mutation on the fusion protein. Accordingly,we sought to exploit the cold sensitivity exhibited by strainscontaining a prlF gain-of-function mutation. In this regard,sohA2 was particularly valuable since the cold sensitivity ofthis mutant is more pronounced.

Isolation and characterization of suppressor strains. Toisolate cold-resistant revertants, we used strain WBS16,which contains the lamB-lacZ fusion, sohA2, and non::TnJO.The fusion protein allowed us to monitor the export pheno-type of the strain on lactose indicator media. Revertantisolation was initially complicated by the tendency of strains

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prlF MUTATIONS INCREASE Lon ACTIVITY 5663

TABLE 1. Bacterial strains and plasmids

Strain or plasmid Genotype or relevant gene carrieda Reference or origin

Bacterial strainsMC4100Pop3186DK2100GP164WBS69SOH4SOH5WBS16SG66SG1117WBS8WBS18WBS19WBS20WBS22WBS23WBS24WBS25WBS42FWBS30SG21097WBS57WBS58WBS59WBS60WBS61WBS62WBS63WBS64SG20843WBS123WBS124WBS125WBS126WBS127WBS128SS14DWBS92WBS93WBS95WBS119WBS120WBS122WBS99WBS100WBS101WBS102WBS115WBS116WBS109WBS145WBS146WBS147WBS148WBS149WBS150WBS151WBS152

PlasmidpLon+

F- araD139 A(argF-lac)U169 rpsL150 relAl flbB5301 ptsF25 deoCl thiMC4100 4(lamB-lacZ)(Hyb42-1) [A pl(209)]Pop3186 prlFlMC4100prlF(Stu)::kanPop3186 prlF(Stu)::kanPop3186 sohAlPop3186 sohA2SOH5 non::TnlOMC4100 CF(lon-lacZ) [A pl(209)]non::TnlOlon::TnlOWBS16 lon-8WBS16 lon-18WBS16 lon-19WBS16 lon-20WBS8 non (Tets)WBS18 non (Tet')WBS19 non (Tet')WBS20 non (Tets)F' 254/WBS22 proC::TnlO recAlll::kanSOH4 argG::TnlOrcsASl::AkanPop3186prlF+ rcsASl::Akan sul zai::TnlOPop3l86prlFl rcsASl::Akan sul zai::TnlOPop3186 sohAl rcsASl::Akan sul zai::TnlOPop3186 sohA2 rcsASl::Akan sul zai::TnlOPop3186prlF+ rcsASl::Akan lon::TnlOPop3l86prlFl rcsASl::Akan lon::TnlOPop3186 sohAl rcsASl::Akan lon::TnlOPop3186 sohA2 rcsASl::Akan lon::TnlOrcsA62 4(cpsBlO-lacZ)SG20843prlF+SG20843 priFlSG20843 sohA2WBS123 lon::TnlOWBS124 lon::TnlOWBS125 lon::TnlOMC4100 lamB14DMC4100 lamBl4D prlF+ rcsASl::AkanMC4100 lamBl4DprlFl rcsAS1::AkanWBS93 lon::TnlOMC4100prlF+ secB::TnSMC4100prlFl secB::TnSWBS120 lon::TnlOMC4100 rcsASl::AkanMC4100prlFl rcsASl::AkanMC4100 sohAl rcsASl::AkanMC4100 sohA2 rcsASl::AkanWBS99 lon::TnlOWBS100 lon::TnlOMC4100 htrA::TnlOWBS99 htrA::TnlOWBS100 htrA::TnlOWBS101 htrA::TnlOWBS102 htrA::TnlOWBS145 4D(lon-lacZ) [X pl(209)]WBS146 (D(lon-lacZ) [X pl(209)]WBS147 '1(lon-lacZ) [A pl(209)]WBS148 4D(lon-lacZ) [X pl(209)]

pBR322 + lon+

26271312This studyG. Phillips; 12G. Phillips; 12This studyLaboratory collectionR. MisraThis studyThis studyThis studyThis studyThis study, select loss TetrThis study, select loss TetrThis study, select loss TetrThis study, select loss TetrThis studyThis studyS. GottesmanThis studyThis studyThis studyThis studyThis studyThis studyThis studyThis studyS. Gottesman; 31This studyThis studyThis studyThis studyThis studyThis studyR. OsborneThis studyThis studyThis studyThis studyThis studyThis studyThis studyThis studyThis studyThis studyThis studyThis studyC. GeorgopoulosThis studyThis studyThis studyThis studyThis studyThis studyThis studyThis study

S. Gottesman; 34

a cF, gene fusion; Hyb, hybrid.

to overproduce a polysaccharide capsule at low tempera-tures (see below). This finding prompted us to use the non

insertion mutation, which is known to block the productionof capsule (reviewed in reference 9). We plated strain

WBS16 on LB plates containing X-Gal and incubated theplates for 48 h at 23°C. Cold-resistant revertants whichconfer either a Lac' or Lac- phenotype were isolated.Calculation of the reversion frequency was complicated by

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5664 SNYDER AND SILHAVY

TABLE 2. Phenotypes of various prlF and lon mutants

PhenotypeStrain genotype Colds/r 1-b Maltose

sensitivity' L sensitivity'

prlF+ r + sprlF::kan r + sprlFl s - rsohAl s - rsohA2 s - rpriFi sohAI,2 su+ r + sprlFl sohAl,2 lon::TnlO r + s

I Scored on LB plates and liquid medium at 23'C (s, little growth; r,wild-type growth).

b The ,B-galactosidase activity of each strain was scored on lactose Mac-Conkey indicator agar (+, red color; -, no color change of the medium).

c Maltose sensitivity (s) is scored as a large zone of growth inhibition in adisk assay (see Fig. 1); maltose resistance (r) indicates viability in maltoseminimal medium and small zones of growth inhibition in the disk assay (seeFig. 1).

the leaky cold sensitivity conferred by sohA2. Revertantsappeared as papillae growing out of a lawn of the parentstrain. We were particularly interested in Lac+ revertantsbecause in these mutants the LamB-LacZ export phenotypeconferred by sohA2 appears to be suppressed.Using P1-mediated generalized transduction, we discov-

ered that 6 of 10 independent cold-resistant Lac+ revertantswere linked to the sohA2 locus. We assumed that thesemutants carryprlF null alleles (see above) and therefore didnot characterize these mutants further. The remaining fourcarried suppressors that were unlinked to priF. A P1 lysateprepared on a pooled population of cells with TnlO insertionsthroughout the chromosome was used to isolate a TnlOinsertion linked by transduction to one of the extragenicsuppressor loci. All four of the extragenic suppressorsisolated showed similar linkage to this TnlO insertion, sug-gesting that all of the suppressor mutations affected the samegene.

Extensive analysis showed that the characteristic pheno-types of priFi, sohAl, and sohA2 lamB-lacZ strains, i.e.,cold sensitivity, Lac-, and maltose resistance, were allsuppressed equally by the four unlinked suppressor muta-tions. These data are summarized in Table 2 and Fig. 1.Since the suppressors have no effect on the Lac activity orthe maltose sensitivity ofprlF+ fusion strains, we concludethat they are involved specifically in blocking the action ofmutant prlFI gene product. The suppressors also restoretemperature sensitivity to temperature-resistant mutantscarrying the htrA (degP) mutation and prlFI, sohAl, orsohA2 (data not shown). This analysis confirms that oursuppressors block all known functions of prlFI (sohA)gain-of-function mutations.The suppressors are Ion null mutations. An Hfr mapping set

(28) was used to map the position of the suppressor-linkedTnJO to a region at approximately 8 to 10 min on thechromosome. Further transductional mapping provided afine-structure genetic map of the suppressor locus (Fig. 2).During the course of transductional mapping, we discoveredthat a lon::TnlO insertion suppressed the cold sensitivity andLac phenotype of the prlFI fusion strain. This findingsuggested that the suppressors may be lon mutations. To testthis possibility, we determined whether known lon muta-tions, such as the TnlO insertion, could confer all suppressorphenotypes and whether the suppressors exhibited pheno-types characteristic of Ion mutants.

14

12

6

4

2

0allele: prlF+ prlFI sohAl sohA2 prlF+ prlFI sohAl sohA2

su 0 0 0 0 + + + +

FIG. 1. Characterization of an extragenic suppressor of sohA2.Maltose sensitivity disk assays were performed as described inMaterials and Methods. The zone of inhibited growth was measuredas (diameter of entire zone) - (diameter of filter paper disk). Thesuppressor (su) allele contained in each strain is wild type (o) orsuppressor (+).

Comparison of the phenotypic effects of our suppressorsand known Ion mutations in mutant and wild-type fusionstrains (Table 2) demonstrates that Ion is indeed a suppressorand that suppression works equally well with all gain-of-function priF mutants. Thus, known lon mutations aresuppressors.One well-characterized Ion mutant phenotype is increased

mutagen sensitivity resulting from a failure of Ion strains torecover from the SOS response (9). We tested our suppres-sor, lon::TnlO, recA, and wild-type strains for mutagensensitivity. Differences in mutagen sensitivity were quanti-tated by using a disk assay with methyl methanesulfonate asthe test mutagen. The lon::TnlO and suppressor strainsexhibited similar sensitivities, while recA and wild-typestrains had greater and lesser sensitivities, respectively (datanot shown). In light of this finding, and because of theobservation that our suppressors cause increased capsuleproduction (see below) in wild-type (non+) strains, weconclude that our suppressors are lon mutations. Moreover,diploid analysis using F' 254 (20), which carries lon+, shows

proc

9 mi

zaJ,

(si

::Tn]O secD tsx

36%11

-.,a

83%'

50%

25%

29%

65%

) 10m

(TnlO) Ion

90%

63%

zba::kan

FIG. 2. Transductional mapping of the suppressor locus. Theapproximate location of suppressor (su) allele mapped with respectto known genetic markers. An arrow points to the selected geneticmarker used in each cross.

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prlF MUTATIONS INCREASE Lon ACTIVITY 5665

that the suppressor phenotypes are recessive. Accordingly,the suppressors must be lon null mutations.Known Lon substrates do not mediate priF function. The

lon gene encodes the well-characterized Lon protease,which globally regulates many physiological responses (for areview of lon genetics, see reference 9). This global regula-tion results from the specific degradation of substrate pro-teins that mediate the various responses. These regulatorysubstrates accumulate in the absence of Lon, and one ofthese could be responsible for suppression in the prlFmutants. Known substrates are involved in filamentation,controlled by the sulA (sfiA) gene product, and capsuleproduction, regulated in part by the rcsA gene product. Twoother substrates for Lon have been inferred from Lon'seffect on the stability of regulatory proteins encoded bybacteriophage X; they are the products of the hflA and hflBgenes. We performed tests of epistasis by using null muta-tions of these genes in strains containing sohA2, the fusionprotein, and the lon suppressor to determine whether re-moval of these known proteins would again allow increasedhybrid protein export. No epistasis was observed (data notshown), indicating that lon-mediated suppression in prlFmutants is not due to the increased level of a known Lonregulatory substrate.

PrIFl increases Lon activity. We reasoned since lon isepistatic to all prlF mutant phenotypes tested, perhaps Lonactivity is regulated by PrlF. This hypothesis was testedgenetically in the following ways.As stated previously, Lon is involved in regulating capsule

production, and this polysaccharide capsule is secreted inresponse to environmental stress (9). The structural genesfor production of the capsule are encoded in the cpsB,operon which is transcriptionally regulated by two activa-tors, the products of the rcsA and rcsB genes (9). RcsB isthought to receive environmental information from the sen-sor encoded by the rcsC gene. RcsB and RcsC showhomology to the activator and sensor components of two-component regulatory systems (9). RcsA, a Lon substrate,interacts with RcsB to activate cpsB transcription (31). Weused a cpsB-lacZ operon fusion to monitor transcription.With use of this fusion, it has been shown that increasingLon activity by increasing gene dosage lowers transcription,while removing lon increases 3-galactosidase activity of thecpsB fusion (34). We observed an approximately 80-foldincrease of 3-galactosidase activity in strains lacking func-tional lon, while prlFl and soh,A2 resulted in a three- tofourfold lower activity (Table 3). This result is consistentwith the hypothesis thatpriFI (sohA) increases Lon activity.To further characterize changes in Lon activity, we stud-

ied the effect ofpriFi on the bacteriophage lysis/lysogenydecision. The lysis/lysogeny decision of is strongly depen-dent on Lon protease activity. The absence of this proteaseleads to decreased levels of cII protein and increased Nprotein (10), resulting in increased lysis by the bacterio-phage. Thus, increased Lon activity should lead to increasedfrequency of lysogenization. Tests of lysogenization fre-quency performed with strains harboring thepriFl mutationshow a 20-fold increase in A lysogenization compared withwild-type strains (Table 4). These results and those pre-sented above support the conclusion that Lon proteaseactivity is increased by the prlF mutations.PriFl increases Lon proteolysis of cytoplasmic precursor

proteins. When protein export is compromised, for exampleby LacZ hybrid jamming, there is a pronounced cytoplasmicaccumulation of the precursor forms of exported proteins.These precursors are abnormal cytoplasmic proteins and

TABLE 3. Evidence that mutations in prlF decrease theP-galactosidase activity of a cpsB-lacZ operon fusion

3-Galactosidase activitybStrain' U (103 (>mol of o-nitrophenol Relative to wild-type

formed/min)/A60 level

prlF+ 1.63 0.19 1.0prlFI 0.55 + 0.17 0.34sohA2 0.39 ± 0.29 0.24prlF+ lon 119 ± 2.5 73prlFl Ion 125 ± 11.8 77sohA2 ito 143 ± 61.5 88

All strains contained the cpsB1O-lacZ operon fusion and the rcsA62mutation.

b Assays for the first three strains were performed four times from twoextracts each; assays for the last three strains were performed two times fromone extract each.

thus likely substrates of Lon (9). Moreover, accumulatedprecursors can interfere with the export reaction by titratingimportant export components. For example, lamB signalsequence mutations cause accumulation of precursor LamBin the cytoplasm, and this in turn inhibits export of otherproteins such as MalE by titrating the secretion-specificchaperone SecB, a condition known as interference (1, 4).PerhapsprlFl ameliorates LacZ hybrid jamming by increas-ing Lon activity to reduce the cytoplasmic accumulation ofharmful precursor proteins.To test the effects ofprlFI on the accumulation of poten-

tially harmful precursors, we used two different test sys-tems. In one, the signal sequence mutation lamB14D (30)was used to internalize precursor LamB; the other usedsecB::TnS to internalize a broader class of wild-type precur-sor proteins (14). We then examined whether priFi and lonwould have any effect on the steady-state level of precursoraccumulation. Figure 3 shows an immunoblot of isogenicstrains containingprlF+, prlFI, and prlFI lon strains carry-ing either a lamB signal sequence mutation (Fig. 3A) or asecB null allele (Fig. 3B). As can be seen, LamB precursors(pLamB) accumulate in the strains containing prlF+ or lon(Fig. 3, lanes 1, 3, 4, and 6). The prlFI lon+ strains,however, show much less accumulated pLamB (lanes 2 and5). This experiment implies that prlFI in the presence of afunctional lon gene can either increase degradation or de-crease synthesis of pLamB. The effect is much less dramaticfor precursor MalE. This result is not unexpected, since it isknown that precursor MalE rapidly assumes a proteaseresistant conformation in the absence of SecB (24).To distinguish between differential synthesis or degrada-

tion caused by prlFI, we used a pulse-chase experiment tomonitor the life of the pLamB14D protein. Four isogenic

TABLE 4. Effects ofprlF and Ion mutations on bacteriophageX lysogenization

Lysogenization frequencyStrain oa Relative to wild-type

level

prlF+ 0.65 ± 0.31 1.0priFI 13.4 ± 4.5 20.6prlF+ lon <0.01 <0.01prlFI lon 1.3 ± 0.9 2.1

aCalculated as number of lysogens per input phage x 100. The experimentwas performed nine times on 3 days as described in Materials and Methods.

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A B

1 2 3 4 5 6pLanB

M. fS _ ~~mLamB~~~~pMaJEmMalE

FIG. 3. Western immunoblot analysis of precursor accumula-tion. Western immunoblot analysis was performed as described inMaterials and Methods. (A) Strains containing the iamB14D signalsequence mutation, the rcsA :1kan allele, and WBS92prlF+ (lane 1),WBS93prlFl (lane 2), and WBS95prlFl and lon (lane 3); (B) strainscontaining the secB::Tn5 mutation and WBS119 prlF+ (lane 4),WBS120prlFl (lane 5), and WBS122prlFl lon (lane 6). The prefixes"p" and "m" denote precusor and mature, respectively.

strains containing prlF+ priFi, and either lon+ or lon werelabeled with [35S]methionine for 30 s and chased for thetimes indicated in Fig. 4 with excess unlabeled methionine.Immunoprecipitation of all samples was performed by usinganti-LamB and anti-MalE antisera prior to electrophoresisand autoradiography as described in Materials and Methods.MalE protein serves as an internal loading control in theseexperiments. The experiment shows that accumulatedpLamB14D is slowly degraded over time in an otherwisewild-type background, causing significant interference towild-type MalE export (Fig. 4A). Inactivation of lon greatlyreduces the degradation and consequently increases theobserved interference (Fig. 4B). Conversely, the priF1 mu-tation results in increased pLamB14D degradation, thereforealleviating interference to MalE export (Fig. 4C). Removalof functional lon from theprlFI strain reduces degradation tolevels observed in theprlF+ lon strain (compare Fig. 4B andD). This result demonstrates that decreased levels ofpLamBobserved in the steady-state experiment shown in Fig. 3 arethe result of increased degradation.We used scanning densitometry to quantitate the degrada-

tion of pLamB14D in various genetic backgrounds. Usingdata from three different pulse-chase experiments, we canshow a reproducible twofold increase in the rate ofpLamB14D degradation in prlFI strains compared withpriF+ strains. Removal of lon reduces degradation in bothpriFI and prlF+ strains to approximately the same rate,which is twofold less than the wild-type rate.Lon activity is increased by an unknown mechanism. The

Lon protease is a heat shock protein and is therefore

A)prIF +l,,n + B)prF + IonV I.A -P .1

)PrIFI Ion T D,)pruIF Ion

30 1' 3' 4' 5- 6- 71 30" 1' 2 3 4 5 6 7_

FIG. 4. Pulse-chase analysis of LamB14D and MalE. LamB and.MalE were pulse-labeled, chased for the indicated times, and thenimmunoprecipitated as described in Materials and Methods. Allstrains contained the iamB14D signal sequence mutation and theindicatedprlF and lon alleles. (A) WBS92; (B) WBS94; (C) WBS93;(D) WBS95.

A)pr/F +37'C B)prIFl 37°C C)prlF+42°C (15') D)Mon,ml nm I 1. < e. -,- -.. -. -. -%.. nAt*. - AAi

F -[ Lon

l * I _ I | x~~~~~~~~O pF_l__|| >~~~~~~~~~~~OmnpA

FIG. 5. Pulse-chase analysis of Lon. Lon and OmpF were pulse-labeled, chased for the indicated times, and then immunoprecipi-tated as described in Materials and Methods. OmpF served as aninternal loading control. Our OmpF antiserum cross-reacts to somedegree with OmpA. Strain genotypes are indicated. (A) WBS92; (B)WBS93; (C) WBS92; (D) WBS95.

transcriptionally regulated by changes in temperature (8, 23).Transcriptional regulation of the classic heat shock regulonis controlled by the alternate sigma factor, &2, the productof the htpR (rpoH) gene (reviewed in reference 21). We wereinterested in determining whether the prlFl gene productwas involved in activating the expression of the Lon gene. Alon-lacZ operon fusion strain was used to show that priF1has no effect on the transcriptional regulation of the lon gene(data not shown). Posttranscriptional regulation of Ion bypriFI was tested by pulse-chase analysis followed by immu-noprecipitation of Lon. As shown in Fig. 5, the prlFImutation has no apparent effect on the synthesis or degra-dation of Lon protein. These results raise the possibility thatthe enzymatic activity of the Lon protease can be activateddirectly.

DISCUSSION

We have used suppressor analysis to identify mutationswhich are epistatic to the function of prlFI. Exploiting theenhanced cold sensitivity of sohA2 mutants, we isolatedcold-resistant Lac' revertants. The only extragenic suppres-sors isolated from this screen are lon null mutations, sug-gesting to us that proteolysis could be involved in producingthe prlFI mutant phenotype. Examination of Lon protease-dependent degradation by two different assays suggests thatLon activity is increased. Further examination of Lon-dependent degradation of export precursors reveals thatprecursor accumulation under conditions of inhibited exportis greatly reduced byprlFl. We have shown that reductionof LamB precursors is due to enhanced degradation of thissubstrate. The rate of pLamB14D degradation is acceleratedtwofold byprlFl.The various phenotypes of priFI, sohAl, and sohA2

mutants appear to be the result of increased Lon activity. Ahigh-copy-number plasmid that increases cellular Lon bygene dosage 30- to 50-fold can make cells cold sensitive,temperature sensitive, and auxotrophic (9a, 29a). Cells con-taining the LamB-LacZ fusion protein become Lac- whenoverexpressing Lon from the plasmid, providing evidencethat increased Lon activity is necessary and sufficient toexplain the prlF mutant phenotypes (29a). However, theplasmid greatly increases Lon synthesis, thus leading to alarge increase in Lon activity, whileprlFl seems to increaseactivity by a more moderate amount. Construction of aplasmid containing the lon gene regulated by a heterologouspromoter, to modestly increase Lon synthesis, may confirmour prediction.Two different experiments have been used to address the

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prlF MUTATIONS INCREASE Lon ACTIVITY 5667

cause of the increased Lon activity. The only known in vivomechanism of increasing Lon activity is to increase synthe-sis. To examine synthesis of Lon, we assayed a lon-lacZoperon fusion and used a pulse-chase experiment. Assumingthat the sensitivities of our assays are sufficient to observeany minor changes, as can be argued from the expectedincrease in Lon levels observed after a 15-min heat shock,we can conclude that synthesis and degradation of Lon arenot affected byprlFl. Therefore, it must be thatprlFI worksdirectly or indirectly to stimulate the activity of the enzyme.This type of activation has never been observed for Lon invivo. It is known that Lon is allosterically activated by ATPconcentration in vitro (18, 19). A shift in conformationalequilibrium toward a high ATP conformation catalyzeddirectly or indirectly by PrlF1 could make Lon more activeat physiological ATP concentrations. Additional work isneeded to clarify the nature of the increased proteolyticactivity.Degradation of the LamB-LacZ fusion protein is not

affected byprlFl. We have used SDS-PAGE to examine theamount of steady-state hybrid protein found in prlFI andwild-type fusion containing strains. We have never observeda decrease in the steady-state levels of hybrid protein in anyof the mutants compared with the wild type (13, 29a). Tofurther address this issue, a pulse-chase experiment con-firmed that the rate of degradation of the hybrid protein isnot changed byprlFI (29a). Therefore, simple degradation ofthe hybrid cannot account for the relief of hybrid proteinjamming. We believe that degradation occurs only withproteins that are retained in the cytoplasm.We propose a model for increased hybrid protein export in

prlF mutants that invokes enhanced precursor degradationas the means of allowing export to continue during unfavor-able conditions. During growth conditions in which synthe-sis of hybrid protein is repressed, export occurs normallybecause export components are free to carry out theiressential function. However, high-level synthesis of thehybrid protein causes a block to the export machinery (13).This generalized export inhibition causes accumulation ofmany precursors in the cytoplasm which could titrate essen-tial export components, thus resulting in lethality. Once thisoccurs, if no mechanism exists to remove the nonproductiveprecursors, export will never resume. Activation of Lon byprlFl alleviates the lethality of hybrid induction by counter-acting precursor accumulation. As long as synthesis andexport can function in the absence of accumulated precur-sors and hybrids, lethality will never occur. We reason thata chaperone may be essential and limiting under conditionsof hybrid jamming because overproduction of the chaper-ones GroEL and DnaK can increase hybrid export in muchthe same way as does prlFI (22).While it is clear that prlFI increases cytoplasmic proteo-

lysis, it remains unclear how the prlF (sohA) mutationssuppress the lethality of degP (htrA) mutants. It has beenargued that the lethality of degP (htrA) mutants is due to theinability to remove toxic proteins which accumulate in theenvelope at elevated temperature (33). One group reportsthat an outer membrane defect can suppress degP (htrA)mutants by allowing leakage of proteins from the cellularenvelope (32). Therefore, the sohA (priF) suppressors couldremove proteins from the envelope by increasing degrada-tion. However, prlFI-, sohAl-, or sohA2-mediated suppres-sion of degP (htrA) temperature sensitivity is Lon depen-dent. Either increased activity of a cytoplasmic protease cancompensate for the absence of the periplasmic protease orLon is an activator of a periplasmic protease. SohB, a

putative periplasmic protease, when expressed from a high-copy-number plasmid can suppress the degP (htrA) mutants,making SohB an attractive candidate for mediating periplas-mic proteolysis in this system (3).The physiological purpose of PrlF remains unclear. Null

mutants of prlF have no obvious phenotypes or growthdefects under standard laboratory conditions. Since expres-sion from the prlF promoter in the presence of a wild-typeprlF gene is extremely low, we consider the wild-typesituation to be very similar to having nopriF gene at all. Allthe knownprlF (sohA) mutations allow increased expressionof the prlF promoter as assayed by lacZ operon fusions intrans, arguing that prlF is autoregulatory (12, 29a). In fact,overproduction of wild-type PrlF results in a phenotypesimilar to that ofprlFI mutants (12). We have not observedany growth conditions which increase the activity of the prlFpromoter in a wild-type background. We speculate thatprlFexists for the purpose of increasing Lon activity and thatincreased synthesis of wild-type PrlF will provide the cellswith some type of adaptive advantage under the appropriateconditions. Another possibility is that PrlF is modified, andnot derepressed, to mediate its function, and simple over-production can shift the equilibrium to produce enough ofthe active form that specific modification is unnecessary.This qualitative versus quantitative model will need to beclarified in order to understand the true mechanism ofwild-type PrlF function.

ACKNOWLEDGMENTS

We thank Susan Gottesman for many insightful discussions andfor providing numerous strains and antibodies. We appreciate thehelpful conversations with Costa Georgopoulos, Michael Maurizi,and Gregory Phillips for clarifying the many different approaches tothis project. As always, this work would not have been possiblewithout help from the entire Silhavy laboratory.

This work was supported by a National Institutes of Health grantto T.J.S. and a Public Health Service training grant to W.B.S.

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