Vol. 164, No. 3JOURNAL OF BACTERIOLOGY, Dec. 1985, p. 1124-11350021-9193/85/121124-12$02.00/0Copyright C 1985, American Society for Microbiology

Insertional Mutagenesis of the lon Gene in Escherichia coli:lon Is Dispensable

MICHAEL R. MAURIZI,* PATSY TRISLER, AND SUSAN GOTTESMANLaboratory of Molecular Biology, National Cancer Institute, Bethesda, Maryland 20892

Received 20 June 1985/Accepted 12 September 1985

The Ion gene of Escherichia coli codes for an ATP-dependent protease. Mutations in lon cause a defect in theintracellular degradation of abnormal and mutant proteins and lead to a number of phenotypic changes, suchas UV sensitivity and overproduction of capsular polysaccharide. We have isolated A transducing phagecarrying the lon gene and used the Ion phage as a target for insertional mutagenesis by a defective transposonTnlO to produce lon::A16A17TnJO derivatives. The A16A17Tn1O (hereafter called ATn1O) elements wereinserted at sites throughout the lon gene and disrupted the coding region between 15 and 75% of the distancefrom the amino-terminal end. Radioactive labeling of proteins in vivo in cells infected with different Alon::ATnJO phage demonstrated that the insertions resulted in the synthesis of truncated Lon proteins. Thelon::ATn1O mutations, when crossed from the phage into the bacterial chromosome, abolished the synthesis ofintact Lon protein, as assayed by antibody on Western blots. An analysis of the protein-degradative ability oflon::ATnJO cells suggests that although the insertions in Ion caused a reduction in ATP-dependent proteindegradation, they did not completely eliminate such degradation either in vivo or in vitro. The lon::ATnJOmutations and a Ion deletion retaining only the amino-terminal 25% of the gene did not affect theenergy-dependent degradation of proteins during starvation and led to only a 40 to 60% reduction in theATP-dependent degradation of canavanine-containing proteins and puromycyl peptides. Our data provideclear evidence that energy-dependent proteolytic enzymes other than Lon exist in E. coli.

The ATP-dependent proteolytic activity in Escherichiacoli both in vivo and in vitro has been attributed to the longene product (3, 4, 12). Ion mutants are deficient in ATP-dependent proteolysis and simultaneously become sensitiveto UV damage and overproduce capsular polysaccharide(16, 18, 29). We have previously demonstrated that the UVsensitivity of lon cells can be explained by the absence of aproteolytic activity which degrades SulA, the UV-inducibleinhibitor of cell septation (23). In vitro studies of ATP-dependent proteolysis with casein and globin as substratesdemonstrate that the major ATP-dependent activity in ex-tracts of E. coli cells is the Lon protease, a 94-kilodalton(kDa) protein called protease La by Goldberg (12). Cellscarrying Ion mutations, such as lon-9 (capR9), make aprotein of the wild-type size but with altered physicalproperties and decreased ability to carry out ATP-dependentproteolysis in vitro (3-5, 39).

Is Lon responsible for all energy-dependent protein deg-radation in E. coli? Do other proteolytic activities exist thatact on the same proteins degraded by Lon? Previous studieshave not provided definitive answers to these questions, inpart because null mutations in lon have not been available. Anumber of observations seem to suggest that proteolyticsystems with some of the properties attributed to Lon maybe present in E. coli cells. (i) The lon mutants analyzed thusfar show no change in the energy-dependent degradation ofk proteins such as 0 and Xis (14, 37; S. Wickner, personalcommunication; S. Gottesman, unpublished observations).(ii) SulA and A N protein, which are stabilized in lon cells(14, 23), are still degraded at a rate significantly above thatfound for other, stable E. coli proteins (25). (iii) T4 infectionof E. coli or transformation of E. coli with plasmids carryingthe T4 function pin stabilizes nonsense fragments,

* Corresponding author.

puromycyl peptides, and canavanine-containing proteins(32, 33). The stabilization is additive with the effects of lonmutations in the same strains (30). (iv) Starvation-inducedproteolysis, which is ATP-dependent, is not affected byclassical Ion mutations (13; M. R. Maurizi, unpublishedobservations). (v) Cells with mutations in the regulator ofheat shock, htpR, are more defective in the proteolysis ofabnormal proteins (such as the ,B-galactosidase nonsensefragment X90 and a temperature-sensitive mutant protein ofthe transcription factor sigma) than are lon mutants (1, 10).

Interpretation of many of these results has depended onthe assumption that Lon activity is in fact totally absent fromstrains carrying Ion mutations. If lon encodes an essential E.coli function, all available mutations may represent leakyalleles of the gene. In fact, ATP-activated proteolysis hasbeen found by Murakami et al. (26) in lon strains. To removethis possible source of ambiguity and to determine whetherlon is an essential gene, we have isolated insertions in the longene carried on a k transducing phage, characterized theinsertions on the phage, and recombined the insertion mu-tations into the host chromosome. Analysis of the resultingIon: :A16A17TnJO strains suggests that (i) lon encodes adispensable E. coli function and (ii) other ATP-dependentproteases that at least partially overlap the substrate speci-ficities of lon exist in E. coli.

MATERIALS AND METHODS

Strains and strain construction. The bacterial strains usedin the experiments in this paper are described in Table 1. P1transduction procedures and screening for lon were done aspreviously described (22, 34). Selection for tetracyclineresistance (Tet) was done on either LB or glucose-M56 agarplates containing 15 ,ug of tetracycline per ml.

Bacteriophage strains. K lon+ phage were selected from alysate of random 5- to 7-kilobase (kb) E. coli Sau3A frag-ments cloned in the BamHI site of int in vector K D69 as

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TABLE 1. Bacterial strains

E. coli Relevant genotype Source of referencestrain

C600 F- thr leu supE tonA NIH"N99 F- galK rpsL NIHN150 F- thr leu supE tonA (21 gp) NIHN5118 F- his Ion-100 rhoHDF026 arg::TnJO rpsL 31RB132 F-A lac rpsL gal zxx::TnJOA&4HH104(pNK217) 21SA431 F- his [X c1857 A(Q-chlA)443] 17SG1041 F- lon-100 Alac 34SG1090 F- lac lon-100 zba-1091::A&TnlO SG1041 + P1(SG20648)SG1091 F- Alac lon' zba-1091::ATNIO SG1041 + P1(SG20648)SG1094 K-12 rel+ (X) WG1 from B. BachmannSG1095 lon-146::ATnJO (X) SG1094 + P1(SG20322)SG1096 Ion-244::ATnIO (X) SG1094 + P1(SG20323)SG1097 lon-230::ATnIO (A) SG1094 + P1(SG21026)SG1098 Ion-224::ATnIO (X) SG1094 + P1(SG20319)SG1110 Alon-510 zba-1091::ATnJO (X) SG1094 + P1(SG4145)SG4038 proCYA221 A(gal-pgl)324 thi rpsL lac 14SG4144 Alon-510 N99 + Xlonl c1857 Alon-510SG4145 Alon-510 zba-1091::ATnJO SG4144 + X lon+2 c1857 zba-

1091: :ATnlOSG20180 cps-1J::lac-Mu dl Ion+ 34SG20250 AlacU169 15SG20252 AlacUl69 Ion-100 zba-300::TnlO 34SG20319 cps-Il::Iac-Mu dl Ion-224::ATnlO SG20180 + Xlon2 c1857 Ion-224::ATnJOSG20322 cps-ll::Iac-Mu dl Ion-146::ATnlO SG20180 + Xlonl cI857 Ion-146::ATnlOSG20323 cps-lI::Iac-Mu dl Ion-244::A&TnJO SG20180 + Xlon2 c1857 Ion-244::ATnIOSG20579 cpsBJO::Iac (imm') proC zaj403::TnIO 34SG20580 cpsBJO::Iac (imm') proC SG20579, Tets selected on FCT agarSG20581 cpsBlO::Iac (imm') Ion-l00 34SG20595 cps-ll::Iac-Mu dl Ion-100 X bio956 c1857 AH1 15SG20648 cpsBJO::Iac (imm') pro' Ion' zba-1091::ATnIO SG20580 + P1(ATnJO inserts from X

Ion)SG20681 cps-ll::Iac Mu dl lon+ X bio956 c1857 AH1 As SG20600 (15)SG21026 cps-Il::Iac-Mu dl Ion-230::ATnlO SG20180 + Xlon2 c1857 Ion-230::ATnIOSG13189 F- his pyrD Agal rpsL Ion-146::A&TnIO SM32 + P1(SG20322)SG13624 F- his pyrD Agal rpsL lon-146::ATnIO A cI ind SG13189 + A cI indSG13636 F- his pyrD Agal rpsL Ion-146::ATnJO (X c1857 ninS) SG13189 + A c1857 nin5SM32 F- his pyrD Ion-100 Agal rpsL 24

a NIH, National Institutes of Health strain collection.

described by Mizusawa and Ward (24). The recombinantlysate described in their paper was the source of both Ximm21 lon+l (K lon'1) and X imm21 lon+2 (K lon+2).The imm21 region in X D69 and the Ion transducing phage

was exchanged for immX either by recombining the transduc-ing phage with X cI857 att8O h80 or by growing the imm21phage in strain SA431, which carries a defective cI857prophage. In both cases, imm' recombinants were selectedon the imm21 lysogen strain N150, which is also resistant toh80 phage. Since both the att80 h80 phage and the defectiveprophage have little or no homology beyond the clone insertsite, recombinants never lose the insert. Other derivatives ofthese transducing phages are described in the text.TnlO mutagenesis. Independent lysates of the Ion

transducing phage Xlon+1 c1857 and Alon+2 cI857 weregrown on the defective TnJO transposon (A16A17TnJO) do-nor strain RB132 (21). (The defective transposon carries thetetracycline resistance gene, but is deleted for transposaseand most of the ISIO elements beyond the outer 70 base pairs(7); it is defective for transposition, but can be comple-mented in trans by Tn.O-coded transposase. In this paper,the defective transposon will be designated ATnJO.) StrainRB132 carries ATnJO on a plasmid and a "high-hopper"TnWO insertion in the chromosome, which provides highlevels of transposase (7). Approximately 1 of 106 of the phagefrom these lysates carried ATnJO inserts. Phage carrying theinserts were detected as plaques on tetracycline-sensitive

hosts (0.5 ml of cells per plate) on low-tetracycline LB agarplates (7 instead of the usual 15 ,ug of tetracycline per ml) oras tetracycline-resistant lysogens.

Selection of tetracycline-resistant lysogens was done instrain SG20595, which carries a cps::iac fusion, a lon-100mutation, and a defective temperature-inducible prophage(15). The cps::iac fusion is negatively regulated by Ion;therefore the lon- cps::iac strain is Lac' (34). K Ion'lysogens of strain SG20595 should be Lac-. Lysogenizationof strain SG20595, which is int+ xis+, is very efficient afterthe cells are heated at 42°C for 15 min to transientlyinactivate the cI857 repressor and allow synthesis of Int. Thelysogens can be screened on lactose indicator plates forexpression of cps: :iac because Ion' transducing phagelysogens will give Lac- colonies, whereas lon- lysogens willremain Lac'.

Strain SG20595 was grown at 32°C in TBMM with biotin(tryptone, 5 g/liter; B1, 1 p.g/ml; maltose, 0.2%; MgSO4, 0.01M; biotin, 30 ,ug/ml) to mid-logarithmic phase, induced for 15min at 42°C, and adsorbed at 32°C for 60 min with the klon+1or Alon+2 lysate which had been mutagenized with ATnJO.The culture was plated on either LB-tetracycline plates (15,ug of tetracycline per ml) or lactose-MacConkey-tetracycline plates. The tetracycline-resistant colonies thatremained Lac' were expected to contain a lon-phagelysogen. Induction of phage from either Lac' or Lac-lysogens gave both tetracycline-resistant and tetracycline-

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sensitive phage. The tetracycline-sensitive phage were alsoint+, as determined by the red plaque assay (6), and hadtherefore lost the insert. These phage apparently were ex-cised by recombination with the defective prophage in strainSG20595 rather than by site-specific recombination at theattachment site. Purification of tetracycline-resistant phagefrom the Lac' lysogens yielded the lon: :ATnJO mutantsdescribed in the text. The four isolates described were fromindependently mutagenized lysates of Xlon+1 c1857 andXlon+2 c1857.

Selection of ATnJO insertions near but not in Ion. Tetracy-cline-resistant lysogens of strain SG20595 were pooled andinduced. At least 50% of the resulting phage were tetracy-cline-resistant and presumably carried a variety of randomATnJO insertions, both in phage DNA and the bacterialinsert. Tetracycline-resistant lysogens of strain SG20250were selected at 32°C, and temperature-resistant, tetracy-cline-resistant recombinants were selected on LB-tetracycline agar. These colonies were pooled, and a P1lysate was prepared on the pooled cells. The lysate was usedto transduce strain SG20580 (proC Ion' cps::iac), selectingfor Pro' Tetr recombinants. These were screened to confirmthat they remained Ion' (Lac-). Subsequent P1 transduc-tions demonstrated that these ATnJO insertions (zba-1091,zba-1092, and zba-1093) were quite close to lon (greater than90% cotransduction).

Recombination of insertion from chromosome to phage. Thezba-1091:::ATnJO insertion was crossed from the chromo-some to the Iion transducing phage by growing the phage onthe tetracycline-resistant host and screening the resultinglysate on low-tetracycline plates for tetracycline-resistantplaques, which arose at a frequency of 10-8. Restrictionanalysis of the resulting phage demonstrated the presence ofan insertion just beyond the C terminus of the lon gene (seeFig. 1 for map of phage).

ion::ATnJO insertion mutations were crossed from thechromosome to the transducing phage in a similar manner.Because we observed that lon::ATnJO strains grew poorly onLB agar, we selected tetracycline-resistant colonies on glu-cose-minimal agar plates containing 15 ,ug of tetracycline perml. The tendency of lon strains to die when grown oncomplex media has been observed previously (9).

Protein synthesis and turnover in phage-infected cells. Cellswere grown, irradiated with UV light, and infected withphage carrying cloned lon genes (multiplicity of infection,10) essentially as described by Mizusawa and Gottesman(23). Infected cells were suspended in 1 ml of growthmedium and labeled with 20 ,uCi of L-[355]methionine (>750Ci/mmol) for the times indicated, and 0.8 ml of suspensionwas mixed with 0.2 ml of 25% trichloroacetic acid(TCA)-0.5% L-methionine. After being washed in acetone,the protein precipitate was dissolved in 0.1 ml of 2% sodiumdodecyl sulfate (SDS) in the upper gel buffer, and 20 ,ul wasrun on a 12% acrylamide gel with the buffers described byLaemmli (20). After electrophoresis, gels were fixed for 2 hin 20% methanol-10% acetic acid, washed for 30 min inwater with 5% glycerol, soaked in Autofluor (NationalDiagnostics) for 1 to 2 h, dried, and placed at -80°C withKodak XAR-2 X-ray film.

Degradation of SulA protein in phage-infected cells wasmeasured by infection with X sulA+ and labeling as above,except that 2 min after the addition of L-[35S]methionine,excess nonradioactive L-methionine (1.5 mM) was added toprevent further incorporation of radioactivity, and severalsamples were taken over a 40-min period. After autoradiog-raphy, the amount of labeled protein was quantitated by

densitometry and by cutting out the radioactive bands fromthe gel, dissolving in NCS tissue solubilizer (New EnglandNuclear), and counting.Immunochemical detection of Lon protease on SDS gels.

Extracts of exponentially growing cells were made by mixingcells with 1/9 volume of 50% TCA, washing the precipitatedprotein with acetone, and running the protein on SDS gels asdescribed above. Proteins were transferred after electropho-resis to nitrocellulose paper (0.45 ,Lm; Schleicher & Schuell)overnight at 30 V in a Bio-Rad Transblot apparatus withTris-glycine buffer in 10% methanol (2). Nitrocellulosesheets were washed with 0.5% bovine serum albumin inphosphate-buffered saline and incubated with anti-Lon pro-tease antibody in this solution for 30 min. Rabbit antibodyraised against purified Lon protease was a gift from L.Waxman and A. L. Goldberg. After being washed threetimes, the nitrocellulose was incubated with goat anti-rabbitimmunoglobulin G (IgG) antibody conjugated to horseradishperoxidase (Capell Laboratories) for 30 min. After beingwashed three times, the sheets were incubated with 50 ml ofphosphate-buffered saline containing 0.4% H202 and 0.1%4-chloro-1-naphthol (added in 10 ml of methanol). After 5min, the sheets were washed with water and stored in thedark at 4°C until photographed.Measurement of protein degradation in vivo. Turnover of

puromycyl peptides was determined as described by Gold-berg (11). Cells were grown in a minimal salts medium with27 mM glucose and 20 mM ammonia. In mid-exponentialphase (A650, 0.3 to 0.6), cells were treated with 80 pug ofpuromycin per ml for 10 min and then for another 10 min inthe presence of 0.2 to 0.5 ,uCi of L-[3H]leucine (2 ,uM) per ml.Cells were washed and suspended in the same mediumwithout puromycin but with 1.5 mM nonradioactive L-leucine and shaken at 37°C. Samples were taken and treatedwith 5% TCA, and the amount of acid-soluble radioactivitywas determined. Degradation of canavanine-containing pro-teins was measured in the same manner except that the cells(arginine auxotrophs) were treated with 50 ,ug of canavanineper ml during labeling with L-[3H]leucine and were chased inthe presence of cold L-leucine and 1.2 mM arginine. Thearginine auxotrophs used were tetracycline-sensitive dele-tion derivatives of an arg::TnJO mutation introduced intostrain SG1094 and its lon- derivatives by P1 transduction.Turnover of protein was measured in ammonia-starved cellsafter growth for two to three generations on L-[3H]leucine.Cells were washed and suspended in fresh medium withoutL-[3H]leucine and ammonia but with 1.5 mM L-leucine. In allcases, acid-soluble radioactivity was measured after the cellswere mixed with 5% TCA and 200 p.g of bovine serumalbumin per ml (final concentrations) and left on ice for 1 to3 h. After centrifugation, 0.5 ml of the supernatant solutionwas counted in 10 ml of Aquasol.

In vitro deletion of lon and transfer of lon deletion to thechromosome. DNA from Alon+1 c1857 was digested withEcoRI and SphI (see Fig. 1). DNA was separated on a 1%agarose gel, and the 3.6-kilobase-pair (kbp) piece containingthe Ion gene was isolated and ligated to pBR322 DNA cutwith the same enzymes. After overnight incubation at 12°Cwith T4 DNA ligase, the ligation mixture was used totransform CaCl2-treated E. coli C600 cells. The desiredplasmid, lacking tetracycline resistance, was found andproved to be a pBR322 derivative in which the EcoRI-SphIfragment of the tetracycline resistance gene was replaced bythe 3.6- kbp Ion' DNA (plasmid plon+500). The purifiedplasmid was cut with PstI and religated, and the DNA wasused to transform lon- strain SG20851. Ampicillin-resistant

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B R ri w P SA Ion+1 0-. I s

Ion

R.HB. R BI '. ..A

A lon+2Ia- 2 Kbp -.1

244 146224

FIG. 1. Schematic of the structure of Alon+1 (top) and Alon+2 (bottom). The site of insertion, size, and orientation of the independentlyisolated X Ion derivatives were determined from restriction enzyme digests with BamHI (B), EcoRI (R), PstI (P), SphI (S), and HindIII (H),alone and in combination. The site of insertion is the BamHI site within the int gene in X D69 (24). The small circles indicate the sites ofinsertion of ATnJO, and the small arrows within the circles indicate the direction of transcription of the tetracycline resistance repressor ofATnJO (38).

transformants that failed to complement the lon mutation(screened by inability to suppress expression of ,B-galactosidase in a cps-lac fusion background [34]) wereanalyzed, and eight of eight were found to have lost 2 kbp ofE. coli DNA containing 75% of the carboxy-terminal end ofIon. This plasmid was designated pAlon-510.The Ion deletion was transferred from pAlon-510 to the Ion

transducing phage and subsequently to the host chromo-some. Xlon+1 was grown on a host containing the pAlon-510plasmid, and the lysate was used to infect strain N99.Ampicillin-resistant transductants were selected, and phagereleased from these cells were collected. These phage hadundergone at least a single recombination with the plasmid toacquire the bla gene of the plasmid. Since X makes turbidplaques on Ion' hosts and clear plaques on lon- hosts (35)and this function can be complemented by the Ion+ gene inthe phage, the lysate could be screened by plaque morphol-ogy and was found to be enriched for X lon- phage (clearplaques). The loss of Lon activity was confirmed by testingthe imm21 phage for complementation of ion-100 in a lysogenof strain SG20595. The imm21 Ion phage was purified, animmX c1857 derivative was constructed, and the phage werelysogenized into the chromosome of strain N99, selecting forimmX at 32°C. The lysogens were transferred to 39°C, and themucoid colonies were purified. These proved to be Ion-:they were sensitive to methyl methanesulfonate (MMS), andboth the mucoid phenotype and MMS sensitivity wereclosely linked to zba-300: :TnlO, known to be linked toclassical Ion mutations. zba-1091 was so closely linked thatwe were unable to select a tetracycline-resistant lon- recom-

binant by P1 transduction. This mutation was introduced bylysogenization with k carrying Ion' and the insertion andthen selection at high temperature for tetracycline-resistantcolonies that remained mucoid (strain SG4145).

RESULTSIsolation of specialized transducing phage carrying lon. A

makes clear plaques on lon strains of E. coli (35), possiblybecause k clI function is less stable in lon- cells than in Ion'cells (14). We postulated that a lambda transducing phagecarrying Ion+ complementing activity would form turbidplaques on a Ion host. Therefore, we searched among alysate of A D69 vector carrying E. coli DNA fragmentspartially digested with Sau3A (24) for a turbid plaque on theIon host strain N5118. Two independent turbid plaques wereisolated, called Xlon+1 and Xlon+2. Single-copy lysogens ofeither of these phage in Ion cells complemented three dif-ferent Ion alleles for capsular polysaccharide synthesis andMMS sensitivity, and therefore they carried a functional Iongene. A c1857 derivatives of the original imm21 phage werethen isolated (see Materials and Methods), and a restrictionenzyme map was determined. The BamHI, EcoRI, andHindlIl restriction sites (Fig. 1) suggested that the two phagecarried the Ion' fragment in opposite orientations. Ourrestriction maps agreed with that previously published bySchoemaker and Markovitz (28) for a 3.0-kb EcoRI-PstIfragment containing lon+ and with sequence information onthe amino-terminal part of the Ion gene (8; S. Goff, personalcommunication). Xlon+1 carries about 0.2 kbp in front of theEcoRI site which begins the Schoemaker and Markovitz

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fragment. Xlon+2 lacks the EcoRI site and about 0.2 kbp ofDNA past this site but carries the intact Ion+ gene (seebelow).We examined expression of proteins from immune and

nonimmune cells infected with Xlon+1 c1857 and Xlon+2c1857. The results (Fig. 2A) show that a 94-kDa protein wassynthesized in very low amounts in immune cells infectedwith either phage. The size of this protein was the same afterinfection with either phage and the same as that found for theion gene product (3, 4). In nonimmune cells (Fig. 2B),synthesis of Ion protein from Xlon+2 c1857 was considerablyhigher, as expected if expression of Lon is under control ofthe lambda PL promoter. When a cIts857 lysogen wasinfected with Xlon+2 cI857, Lon was expressed at low levelsat 32°C but at high levels after heat inactivation of cI at 42°C,consistent with transcription from PL (Fig. 2C). In the same

TABLE 2. Complementation screening for ion insertionmutations

lon allelel Prophage Lac b 3-Galactosidase (U)"lon-100 None + 14lon+ None - 1.7Ion-100 X lon+ - 1.4Ion-100 X lon::ATnJO + 15Ion+ X lon::ATnJO - 2.4

a All host strains carried cps-II::Iac operon fusions and a defective Xprophage, bio939 int+ xis+ A(SaI-Xho) c1857 AH1. The Ion' host was strainSG20595; the lon- host was strain SG20681.

b Determined on lactose-MacConkey indicator agar at 32°C.c Cells were grown in glucose-minimal medium at 32°C and assayed for 1-

galactosidase specific activity, expressed in Miller units (22).

B Non-Lysogen

*w :.&*40.,No*0.:4

_

o-n

FIG. 2. Lon protease synthesis from Alon'c1857. Autoradiograms were made of L-[35S]metteins separated on 12% acrylamide gels run inlabeled for 20 min following infection of UV-irraXlon+1 c1857, Xlon+2 c1857, or control phageInfection of A lysogen strain SG13624, in wproteins to appear should be from the cloned E.active cI protein represses expression of A gerground was caused by insufficient irradiation i

synthesis of proteins from the chromosome. Arnprotease. (B) Infection of E. coli Ion noninSG13189). The proteins made are mostly Xinfecting A DNA (compare with D69 control larat least six proteins made from the inserted E.appearing here are presumably transcribed frorror Pint since they are not seen in high amountshas the insertion in the opposite orientationlysogen (panel A). (C) Infection of E. coli X c

SG13636. Cells were infected at 300C with Aloncontrol), and half of the culture was labeled atwas shifted to 42°C and labeled after 5 min. Arrmprotein.

cells, SulA protein, synthesized from its own promoter, wasmade at high levels at 30°C (Fig. 2C). These data areconsistent with the direction of transcription of lon reported

C AIon*2 in by Schoemaker and Markovitz (28). Expression of lon fromts Lysogen Alon+1 decreases in nonimmune cells, presumably because

4 convergent transcription from the PL promoter interferes300 420 with Ion expression (36).

We believe the lon promoter is present in both the X lon'MONO clones, since lon complementing activity was present in

single-copy repressed lysogens of both phages (see Table 2).It remains possible that lon is expressed at a low level fromthe lambda int promoter in Xlon+2. The poor expression ofIon from its own promoter after infection of heavily UV-irradiated cells may result from the loss of an unstable factor

_jib required for efficient synthesis of Lon.In addition to Lon, several other proteins were detected in

nonimmune cells infected with Alon+2. Because these pro-teins were all synthesized in greater amounts in nonimmunecells, they are apparently transcribed in the same directionas Ion. The 67-kDa protein was presumably the same as that

q_mi observed by Schoemaker and Markovitz (28) after labelingproteins made from a plasmid derivative carrying a DNAfragment including lon. The functions of these proteins arenot known, but they are probably not related to Lon prote-ase since they did not change in any of the Ion insertionmutants (see below). One of our insertions near lon (zba-1091::ATnJO), which retained a normal Lon' phenotype,disrupted the gene for the 67-kDa protein near the carboxy-terminal end and resulted in a truncated protein of about 62

1 c1857 and Alon+2 kDa (data not shown).thionine-labeled pro- Insertion mutagenesis. We used a defective TnJO trans-SDS. Proteins were poson, ATnJO, to isolate insertion mutations in lon carrieddiated Ion cells with on X. The defective transposon (originally designatede X D69 c1857. (A) MA17TnJO), 2.9 kb long, carries the tetracycline resistance/hich the only new gene and the outer ends of the ISiO elements which normally

nes. The high back- flank TnJO, but is deleted for transposase and most of thewith UV to prevent ISJO elements beyond the outer 70 base pairs (7). ATnJO isrow, Position of Lon defective for transposition, but can be complemented innmune cells (strain trans by TnJO-coded transposase. A vector and host haveproteins from the been described which allow the easy isolation of ATnJO

ne). Arrows indicate transpositions from a plasmid to X (7, 21). We developed acoli DNA. Proteins complementation assay to allow easy screening for A

i the X promoters PL lon: :ATnIO insertion mutations. A defective X prophagewith Xlon+l, which carrying attB and the X integration functions Int and Xisor in an infected,1857 lysogen strain under the control of the temperature-inducible c1857 repres-

+2 and A sulA (as a sor was introduced into a strain carrying a Ion mutation and30°C; the other half a cps::iac fusion. The original Ion- strain was Lac', whereasow, Position of SulA a lon+ version of the same strain was Lac- (Table 2), since

Ion regulates the level of expression of genes necessary for

A Lysogen>, xo ", x

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INSERTION MUTATIONS IN lon 1129

a -b c d e B a b cMW

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slo,.-l

23K 4-__w

FIG. 3. (A) Restriction enzyme digests of Xlon2 DNA containing vTnJO inserts. DNA from the different phages was digested with BamHIand EcoRI, run on 1% agarose gels, and stained with ethidium bromide. Lanes: a, Alon+2 DNA digest; b through d, DNA from Xlon2 carryinglon-224::ATnJO, Ion-224::ATnIO, Ion-146::ATnJO, and Ion-230:t:ATnJO, respectively. The arrows show the two new DNA fragments obtainedby insertion upstream or downstream of the BamHI site in the Ion gene. Two new BamHI-EcoRI fragments are formed with each insertbecause ATnJO has an internal EcoRI site 900 base pairs from one end. (B) Autoradiograms of L-[35S]methionine-labeled proteins synthesizedfollowing infection of UV-irradiated cells of strain SG13189 with Alon+2 or Xlon2 with Ion::ATnJO inserts. Proteins from xlon+2 (a), Xlon2Ion-244: :ATnJO (b), and Xlon2 Ion-230: :ATnJO (c) were separated on a 12% acrylamide gel in SDS. In a separate experiment, proteins fromXlon+2 (d), Xlon2 Ion-224::ATnJO (e), and Alon2 Ion-146::ATnJO (f) were separated on a 10% acrylamide gel in SDS. Arrows indicate wild-typeLon protease and truncated Lon proteins. The 25,000-molecular weight (25K) tetR protein was found only in Xlon2 ion-230::ATnJO, whichby restriction analysis was shown to contain an insert in an orientation opposite that of the other inserts.

capsular polysaccharide synthesis, as reflected by expres-sion of the cps::iac fusion (34). Since Ion' is dominant,introduction of a Ion' phage at the attachment site willcomplement the lon-100 mutation to give Lac- colonies,whereas X lon::ATnJO lysogens should not complement itand therefore will remain Lac' (Table 2). Lysogenization ofthe recipient strain SG20595 is very efficient after the cellsare heated for 15 min at 42°C to inactivate the cI857repressor and to allow transient expression of Int (15). Byselecting tetracycline-resistant lysogens of strain SG20595and screening the lysogens for Ion complementation by coloron lactose-MacConkey indicator plates, we could simulta-neously isolate the 1 phage in 106 carrying the ATnJOinsertions and screen for those which inactivated Ion. Of thecolonies isolated on LB-tetracycline plates, approximately5% were found to remain Lac' and therefore failed tocomplement lon-100. A lon+ cps::iac host lysogenized withthe same Ion phage remained Lac- (lon+) (Table 2); there-fore, these insertion mutatiohs are recessive.

Isolation and characterization of the lon: :ATnJO insertionson the transducing phage allowed mutations to be analyzedwithout any assumptions about whether lon encodes an

essential E. coli function. Based on the restriction map of

four independent inserts into the Alon+2 phage (Fig. 1 and3A), our most amino-terminal insertion (ion-230) should beabout 15% of the distance from the N terminus, and our mostcarboxy-terminal insertion (lon-224) should be 75% of thedistance from the N terminus, using the estimates ofSchoemaker and Markovitz (28) for the limits of the gene.The sizes of the proteins made from the transducing phagewere determined as described above after infection ofheavily UV-irradiated nonlysogenic cells (Fig. 3B). Theinserts in all cases abolished synthesis of only the 94-kDaprotein we had previously identified as Lon. In all fourinsertions, a new band of about the mobility expected for a

protein of the size predicted from the position of the inser-tions was observed. In addition, one insertion mutantshowed a band with the mobility of a 25-kDa protein whichwas almost certainly the tetracycline resistance repressor,

synthesized from the lambda PL promoter (38). We haveobserved this band in a number of independent ATnJOinsertions in several different genes cloned in the X D69vector. This protein was seen only when the insertion was

oriented so that transcription of the tetracycline resistancerepressor gene (38) from PL was possible.

Ion is a dispensable gene for E. coli. Having obtained and

A

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0.5-

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1130 MAURIZI ET AL.

I Select tetracycresistant lysog

at 320C

M I QR.7 A Ilon+

A

PLc I857

clinelens

att Ion::Tn10AnII I

Select tetracyclineresistant survivors

at 390C

/on::Tn10_Lon-

characterized ATnJO insertions spanning the lon gene, weattempted to introduce the mutations into the host chromo-some to determine whether Ion is in fact a dispensable geneand, if so, to determine the properties of a cell containingonly small fragments of Lon. The use of the defective TnJOinsertions simplified this task considerably (Fig. 4A). Weselected lysogens of the c1857 derivatives of the lon::ATnJOphage by selecting for tetracycline-resistant colonies at 32°Cafter adsorption of the phage. In the absence of the site-specific integration function Int, the phage lysogenize byhomologous recombination between the bacterial insert onthe phage and the chromosome, at the Ion site. From theselysogens, temperature-resistant, tetracycline-resistant survi-vors were selected. Such survivors were not rare; from 5 to35% of the temperature-resistant survivors were also tetra-cycline resistant, depending on the insertion. Thelysogenized strain (SG20180) carried a cps::lac fusion, al-lowing the detection of lon- cells as Lac' (34). As expected,all tetracycline-resistant, temperature-resistant segregantsbecame Lac' when the original phage carried lon::ATnJO,but not when it carried an insertion elsewhere in the bacterialor phage DNA. P1 grown on these tetracycline-resistantderivatives was used to transduce a variety of strains totetracycline resistance; in all cases, 100% of the transduc-tants acquired all lon phenotypes (MMS sensitivity, capsuleoverproduction, and the ability to support the growth of AOts phage at high temperatures; see below). Transductionfrequencies were not abnormally low, and there was no

evidence that the strains were acquiring secondary muta-tions. We did observe that strains carrying Ion::ATnJOmutations made very small colonies on LB-tetracycline andLB plates. Microscopic examination of these cells showedthat the cells had filamented, a characteristic of lon- strainsafter DNA damage. Introducing sulA mutations into suchstrains blocked SOS-induced filamentation and allowed bet-ter growth on LB agar. Poor growth of Ion strains on richmedium has been observed by others (9); the total loss of lonactivity in these insertion mutants seems to exacerbate thisphenotype.

Insertions in the Ion gene can be recombined from phageto chromosome and back to transducing phage to confirmthat introducing the insertion into the chromosome does notlead to any unexpected rearrangements. One of the inser-tions originally isolated in the Alon+1 c1857 phage wasion-146: :ATnJO. From haploid segregants carrying this mu-tation in the host chromosome (Fig. 4A), we isolated tetra-cycline-resistant, temperature-resistant survivors whichshowed the Ion phenotype and in which lon was 100% linkedto tetracycline resistance in P1 transductions. Using thisstrain as a host, we introduced the Xlon+2 c1857 phage,formed lysogens, and induced the phage to obtain tetracy-cline-resistant derivatives (Fig. 4B). We demonstrated thatthese phage now carried a Ion::ATnJO mutation by using thecomplementation test described above. Finally, the restric-tion enzyme pattern of this Xlon2 lon::ATnJO derivativeshowed that the ATnJO and the new derivative were inserted

Lon+

Lon+ ti i| rrrA-PL CIOD/ MA J

F-- I

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INSERTION MUTATIONS IN Ion 1131

Lon- B

PL

J

Lon+lon::TnlO att AR cI857 PL Ion+~55~5555~ i W -

-

!

Induce phage at 400Cand screen for A carryingtetracycline resistance

att P

PLc1857 A on 2::Tn J

R AFIG. 4. (A) Schematic diagram of the procedures used to cross mutations in Ion from the phage to the E. coli chromosome. The example

shown starts with Alonl lon-146: :ATnJO, in which the Ion gene is oriented opposite to the PL direction of transcription. Cross-hatched boxes,bacterial copy of Ion; open boxes, cloned bacterial DNA; solid boxes, the ATnJO insertion. (B) Transfer of chromosomal lon mutations to Xcarrying the wild-type Ion gene. The example shows transfer of a ATnJO insert to Xlon+2, in which the Ion gene is oriented in the samedirection as transcription from PL. Note opposite orientation of X in panels A and B. Details are given in Materials and Methods.

in the same location relative to lon. The orientation of thetransposon was inverted with respect to PL, as expected.This was confirmed by the high-level synthesis of the tetra-cycline repressor after infection with the Xlon+1 derivativebut not the Xlon+2 derivative. Three other Xlon2 lon::ATnJOmutations were crossed into the chromosome and back ontothe Xlon2 phage; in all cases the restriction enzyme patternof the starting and final phage indicated that no rearrange-ment had occurred during these recombination events.To confirm that the lon::ATnJO mutations did not present

a serious obstacle to cell growth, we carried out P1 transduc-tions in which we could screen for lon: :ATnJO rather thanselecting for it. P1 grown on the pro' lon::ATnJO donorstrains was used to transduce recipient strain SG20580 (Ion+proC cps::iac), selecting either Pro' or Tetr recombinants.Control transductions were carried out with donor DNAcarrying ATnJO close to but not in Ion (see Materials andMethods). The results for three independent lon::/TnJO

insertions indicate that the cotransduction frequencies forproC and Ion were comparable, regardless of whether thelon: :ATnJO was selected directly or screened for, among thePro' recombinants (Table 3, Fig. 5). In addition, we had nodifficulty introducing these lon: :ATnJO mutations into avariety of other E. coli strains by P1 transduction. There-fore, we conclude that E. coli can tolerate TnJO insertionswhich span the lon gene and abolish synthesis of intact Lonprotease.

Assay for Lon protein with anti-Lon protease antibody. Toconfirm that cells carrying the ATnJO insertions lackednative Lon protein, extracts of isogenic Ion', ion-100, andlon::ATnJO cells grown in LB were made by boiling cells inSDS, and the proteins were separated on SDS gels. Westernblots (2) made with antibody against purified Lon proteaseshowed the presence of an intact 94kDa protein in Ion' cells;this protein comigrated with pure Lon protease and also withthe major radioactive band of E. coli protein made during

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1132 MAURIZI ET AL.

TABLE 3. Ion-proC cotransductionCotransduction frequency

Donor strain Selec- (%)b(relevant genotype)a tion

tsx proC Ion TnIO

SG1090 (lon-100 zba-1091::ATnJO) Tetr 42 23 73SG1091 (lon+ zba-1091::ATnJO) Tetr 46 30SG20252 (Ion-100 zba-300::TnlO) Tetr 43 13 76SG20322 (lon-146::ATnJO) Tetr 46 26 100SG20323 (1on-244::ATnlO) Tetr 55 35 100SG1090 (lon-100 zba-1091::ATn]O) Pro' 41 18 17SG1091 (lon+ zba-1091::ATnJO) Pro' 43 - 19SG20252 (lon-100 zba-300::TnlO) Pro' 64 NT 13SG20322 (Ion-146::ATnJO) Pro' 33 NT 15SG20323 (1on-244::ATnJO) Pro' 22 NT 15

a The recipient strain in all cases was SG20580 (proC tsx lon cpsBIO::Iac).Analysis of the segregation pattern for zba-1091, Ion, and tsx suggests thatzba-1091 is clockwise to lon (data not shown).

b NT, Not tested; -, No lon allele in the cross.

infection with Alon+2 (Fig. 6). None of the insertion mutantscontained any intact Lon protease (the limit of sensitivitywas less than 1% of the wild-type level, as determined byserial dilution of extracts). Lower-molecular-weight proteinswere seen in three of the mutants, as expected (Fig. 6, lanesc, e, and f); the sizes (70 kDa for lon-224, 55 kDa for lon-244,and 23 kDa for lon-146) corresponded to those of theproteins made by the respective Ion phages carrying thesealleles (Fig. 3). The 15-kDa protein seen after infection withXlon2 lon-230::ATnJO and expected to be made in lon-230mutant cells was not detected with the antibodies used.These results confirm the disruption of the Ion gene in theinsertion mutants, reveal the presence of the amino-terminalfragments of the disrupted protein, and establish the absenceof any cross-reacting carboxy-terminal fragments of Lonthat might have arisen from restarts. The lon-100 mutantcontained a small amount (3 to 5% of the wild-type level) ofLon protein that appeared to have the same molecularweight as native Lon protease (data not shown).

Partial deletion of the Ion gene on plasmid plon+500 withPstI eliminated >70% of the 3' end of the gene (see Materialsand Methods) (Fig. 1). Transformants with the deletionplasmid (pAlon-510) made a large amount of a 25-kDa proteindetectable in Western blots with anti-Lon protease antibody(Fig. 6, lanes k and 1). Smaller amounts of this protein wereseen in cell extracts of strain SG1110, in which this deletionhad been crossed from the plasmid to the E. coli chromo-some (lane b).

Properties of lon::ATnJO strains. Isogenic strains carryinglon+, lon-100, or the lon-146::ATnJO insertion (which make a23-kDa fragment, about 25% of the wild-type protein size)

0.18 >

0.15 >

0.43 >

proC tsx Ion zba-1091::ATn10

< 0.44

< 0.27

FIG. 5. Cotransduction of lon and proC. Numbers indicatecotransduction frequencies. Arrowheads point away from selectedmarker.

a b c d e f g h i j

94.~70-.

55-.

25-23-.

k I

__0P S~~~- VW,.

.....,__p

_~~~~A0

-I

FIG. 6. Lon cross-reactive material made in lon+ and lon- cells.Cells (1 ml) in late log phase were treated with 5% TCA, and 1/5 ofthe washed protein precipitate was run on a 10% acrylamide gel inSDS. After transfer to nitrocellulose paper, the proteins that boundto anti-Lon protease antibody were visualized with horseradishperoxidase-conjugated goat anti-rabbit IgG. Lanes: extracts ofstrains (a) SG1094 (lon+), (b) SG1110 (Alon-510), (c) SG1098(lon-224), (d) SG1097 (lon-230), (e) SG1096 (lon-244), and (f) SG1095(lon-146); g through j, extracts of strain SG1094 (Ion') undiluted anddiluted 1:3, 1:9, and 1:27, respectively: k and I, extracts fromtransformants carrying plasmid pAlvon-510 (PstI-PstI deletion),strains SG1094 (lon')(pAlon-510) (k) and SG1097 (lon-230)(pAlon-510) (1). Sizes are indicated in kilodaltons.

were constructed and compared for lon characteristics (Ta-ble 4). Overproduction of capsular polysaccharide can bemeasured as the expression of ,-galactosidase from cps::lacfusions (34). The ion-100 mutation increased ,B-galactosidasesynthesis more than 25-fold in this fusion mutant, and thelon-146::ATnJO mutation increased synthesis an additionalfive- to sixfold.The sensitivity of lon- strains to DNA-damaging agents,

believed to be due to accumulation of the SOS-inducible celldivision inhibitor SulA, can be estimated by the efficiency ofplating (EOP) on agar containing MMS (16). Here, as incps::lac expression, lon::ATnJO mutant strains were moredefective than the lon-100 mutant (EOP, 10-5 versus 0.03).Direct examination of the half-life of SulA in these strains bya pulse-chase with L-[35S]methionine indicated that the half-life, 1.2 min in wild-type strains, was about 19 min in lon-100and 33 min in lon-146::A&TnJO hosts. Thus, this normal E. coli

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INSERTION MUTATIONS IN lon 1133

TABLE 4. Properties of lon::ATnJO mutants'

1-Galacto- EOP with SulA DegradativeIon allele sidase MMSC half-life activity

(U)b (min)d (elativ

Ion+ 9.8 0.3 1.2 10-4Ion-100 250 0.01 19 0.4lon-146::A&TnJO 1,456 3 x 10-5 33 0.3lon-244::ATnJO NTf 4 x 10-5 NT 0.4

a Two sets of isogenic strains were constructed. Derivatives of strainSG20580 were used for the P-galactosidase measurements (as an indicator ofcapsular polysaccharide production); derivatives of strain SG4038 were usedfor measurements of MMS sensitivity (as a measure of UV sensitivity) and AcI857 Ots growth (as a measure of degradative activity).

b Cells were grown in glucose-minimal medium at 30'C and assayed for P-galactosidase as described by Miller (22) as a measure ofcpsBO: :lac (capsule)expression.

c Tested on LB agar plates containing 0.025% MMS.d Measured after infection of UV-irradiated strain SG13624 with A c1857

sulA+. Values for lon+ and ion-100 cells are from Mizusawa and Gottesman(23).

' EOP of A c1857 Ots on indicated strain at 42'C compared with that on aIon' host at 32°C. A cI857 Ots plated efficiently (EOP of 0.8 to 1.0) on all fourstrains at 32°C.f NT, Not tested.

protein is stabilized significantly but not completely in hostscarrying insertions in the Ion gene.

Degradation of abnormal proteins is also affected by Ionmutations. A genetic test of this phenotype is the EOP of Aphage carrying a temperature-sensitive mutation in the rep-lication gene 0 (16). We assume that this temperature-sensitive protein becomes particularly susceptible to Lonproteolysis at high temperature. Both the lon-100 mutationand the lon-146::ATnJO mutation were sufficient to allowgrowth of X cI857 Ots at the normally nonpermissive tem-perature of 42°C.Thus, the lon::ATnJO mutation conferred all expected Ion

properties on the host and in some cases appeared to betighter than the ion-100 mutation.

Protein degradation in Ion mutants. A more direct test ofthe ability of the insertion mutants to degrade abnormalproteins was made by pulse-labeling cellular protein withL-[3H]leucine in the presence of puromycin. After treatingcells for 10 min with 80 ,ug of puromycin per ml, 50 to 80%of the protein labeled in a short pulse with radioactiveleucine was abnormal in that it was degraded with anunusually short half-life of 20 to 30 min, whereas mostnormal E. coli proteins are degraded with half-lives of >20 hin growing cells (25). The degradation of puromycyl peptidesin Ion' cells was compared with that in cells containinglon-146::ATnJO (Fig. 7A). The Ion insertion mutant showed a30% reduction in the rate of decay of abnormal proteins. Inall cases the total radioactivity incorporated during the pulsewas the same for wild-type and mutant cells. In otherexperiments, degradation of puromycyl peptides andcanavanine-containing proteins was reduced about 50%o in allfour of the Ion insertion mutants compared with that inwild-type cells (data not shown). The defect in degradationof abnormal proteins in the Ion insertion mutants wascomparable to that reported previously for other lon mutants(31).The residual degradation of abnormal proteins in the

mutant resembled Ion-dependent degradation in being inhib-ited by energy deprivation (brought on by removal of glucoseand addition of KCN; Fig. 7A). Upon removal of the KCNand addition of glucose, the cells immediately began to grow

at their characteristic rate, and protein degradation resumedin both Ion' and lon- cells (Fig. 7A), indicating that, asshown previously by Goldberg (11), the energy deprivationdid not simply kill the cells.

Degradation of normal proteins in wild-type cells averages<1% per h during growth and increases to 4 to 6% per hduring nitrogen starvation (13). Our lon- cells showed ex-actly the same increase in protein degradation as wild-typecells did when nitrogen was removed (Fig. 7B). This degra-dation was partially energy dependent, as indicated by the80% inhibition by KCN. Thus, starvation-inducedproteolysis is truly independent of Lon and requires meta-bolic energy.

DISCUSSION

This paper has described the construction and character-ization of insertion and deletion mutations in Ion, the genefor an ATP-dependent protease in E. coli. Our procedurewas to isolate a lambda transducing phage carrying lon andto mutagenize Ion on the phage by inserting a defective TnJOtransposon. We have thus been able to obtain severalwell-defined mutations in lon. Because they were isolated ina strain diploid for the Ion locus, selection of mutations onthe phage was not biased against abolishing any particularpart of lon or against any mutation that might exert polareffects on adjacent genes. These mutations were subse-quently crossed into the chromosome, replacing the activeIon gene, and the resulting mutants had all the expectedproperties of lon- cells.The Ion gene was cloned in bacteriophage lambda with a

functional assay (turbid plaque growth). Analysis of thephage DNA with restriction enzymes gave results consistentwith those previously obtained with a plasmid clone of lon(28), and the ATnJO inserts were found to disrupt therestriction fragments expected to lie within the lon gene.Protein analysis on SDS gels showed a protein of the sizepreviously identified for Lon protease made by the lon+phage during infection of UV-irradiated cells; this proteinwas absent after infection with phage carrying ATnJO inser-tions in Ion. Subcloning of the putative lon gene from thephage into the multicopy plasmid pBR322 yielded transform-ants with high levels of Lon protease, identified by immu-nochemical and enzymatic assays (data not shown). In vitrodeletion of the Ion gene on this plasmid eliminated the highamounts of Lon protease.The insertion mutations crossed into the chromosome

were 100% linked to lon by P1 transduction and had the samelinkage to proC (20%) as previously shown for Ion (16). A Ionphage with the ATnJO inserted in E. coli DNA downstreamfrom lon was also crossed into the chromosome. Analysis ofthe linkage between Ion, the tetracycline resistance gene,and either proC or tsx suggests that Ion lies between proCand the downstream insertion and is therefore transcribedclockwise on the E. coli chromosome (unpublished data).The Ion gene codes for an ATP-dependent protease (3, 4).

Many of the phenotypes of lon mutants can be explained bythe proteolytic activity of the Lon protease. Thus, lon cellsare partially defective in the degradation of abnormal pro-teins and certain missense proteins, and they fail to rapidlydegrade SulA protein, an inducible inhibitor of cell division.The persistence of SulA in cells leads to irreversiblefilamentation and can explain the UV sensitivity of lon- cells(19, 23). The overproduction of capsular polysaccharide inIon mutants may also be due to a defect in the degradation ofa positive regulator of capsule synthesis (15). While previ-

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C'I)0

x

Ea

zCa

CD1=0

w0w0

0-w0-

0

z0

0

CDwazL

0cEa.-Ja:

0z

TIME (h) TIME (h)FIG. 7. (A) Puromycyl peptide degradation in growing lon- cells. Cellular protein was labeled with L-[3H]leucine in the presence of

puromycin (80 ,ugIml) and then chased with nonradioactive L-leucine in the absence of puromycin. The amount of TCA-soluble radioactivitywas determined. Symbols: 0, 0, Oi, SG1094 (lon'); A, A, A, SG1095 (lon-146); 0, A, complete medium plus glucose; 0, A, glucose removedat the start of the chase and 1 mM KCN added; 3,A, glucose removed and KCN added as above, and at the time shown by the arrow, cellswere washed to remove the KCN and suspended in fresh medium containing glucose. (B) Normal protein turnover during ammonia starvationin lon- cells. Cells were labeled with L-[PH]leucine during exponential growth on 4 mM ammonia. When the cells stopped growing (A590, 0.9)200 jig of L-leucine per ml was added, and acid-soluble radioactivity was determined at 2-h intervals. Solid symbols, complete medium plusglucose; open symbols, glucose removed and 1 mM KCN added at the start of the chase. Strains: 0,0, SG1094 (lon'); A,A, SG1095(lon-146); *O, SG1096 (lon-244); V,V, SG1110 (Alon-510).

ously isolated Ion mutants were defective in the knownfunctions of Ion, none was demonstrated to carry a nullmutation. At least one mutation (lon-9 [capR9]) results in anactive but unstable Lon protease of the same size as thewild-type protein (5, 39). Mutants carrying lon-100, previ-ously thought to be a deletion (16), make a small amount ofLon protein which apppears to be the same size as intactLon protease (data not shown). Southern gel analysis of cellscarrying the ion-100 mutation suggested a change in the sizeof the promoter-proximal restriction fragment (27). lon-100may be a deletion or an insertion in the promoter region ofIon.

Previously, ambiguity concerning the amount of residualLon activity in mutant cells prevented any definitive answersto several important questions, such as whether Lon is an

essential protein and whether all energy-dependent degrada-tion in E. coli is due to Lon protease. Our results provideunequivocal answers to these questions. Ion is not essentialfor the growth of E. coli, since our mutants lacked Lonprotease activity. Our insertions and an in vitro-constructeddeletion disrupted the coding region of the Ion gene at sitesbetween 15 and 75% of the distance from the amino terminusof the protein. These mutations abolished the intact Lonprotease and permitted synthesis of amino-terminal proteinfragments from 15 to 70 kDa in size. It is extremely unlikelythat such fragments, particularly the smaller ones, wouldpossess Lon protease activity. There is no evidence forsynthesis of other Lon fragments either after infection with X

lon: :ATnJO or by Western blot analysis of chromosomallon: :ATnJO mutants.We conclude that the residual protein degradation in our

mutant cells is lon-independent activity. In addition, thisresidual degradative activity of both normal and abnormalproteins is energy dependent or partially so (Fig. 7). Thus, it

is likely that other proteases in E. coli may be ATP depen-dent or have effectors that reflect the energy state of the cell.

ACKNOWLEDGMENTS

We thank Fred Goldberg and Lloyd Waxman for the gift ofanti-Lon antibody. We thank Sankar Adhya and Sue Garges for theircomments on the manuscript.

LITERATURE CITED1. Baker, T. A., A. D. Grossman, and C. A. Gross. 1984. A gene

regulating the heat shock response in E. coli affects proteolysis.Proc. Natl. Acad. Sci. USA 81:6779-6783.

2. Burnette, N. W. 1981. "Western blotting": electrophoretictransfer of proteins from sodium dodecyl sulfate-polyacry-lamide gels to unmodified nitrocellulose and radiographic detec-tion with antibody and radioiodinated protein A. Anal.Biochem. 112:195-203.

3. Charette, M. F., G. W. Henderson, and A. Markovitz. 1981.ATP hydrolysis-dependent protease activity of the lon (capR)protein of Escherichia coli K12. Proc. Natl. Acad. Sci. USA78:4728-4732.

4. Chung, C. H., and A. L. Goldberg. 1981. The product of the lon(capR) gene in Escherichia coli is the ATP-dependent protease,protease La. Proc. Natl. Acad. Sci. USA 78:4931-4935.

5. Chung, C. H., L. Waxman, and A. L. Goldberg. 1983. Studies ofthe protein encoded by the lon mutation, capR9, in Escherichiacoli. J. Biol. Chem. 258:215-221.

6. Enquist, L. W., and R. A. Weisberg. 1977. A genetic analysis ofthe att-int-xis region of coliphage lambda. J. Mol. Biol.111:97-120.

7. Foster, T. J., M. A. Davis, D. E. Roberts, K. Takeshita, and N.Kleckner. 1981. Genetic organization of transposon TnlO. Cell23:201-213.

8. Gayda, R. C., P. E. Stephens, R. Hewick, J. M. Schoemaker,W. J. Dreyer, and A. Markovitz. 1985. Regulatory region of theheat shock-inducible capR (lon) gene: DNA and protein se-

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Second-site mutations in capR (Ion) strains of Escherichia coliK-12 that prevent radiation sensitivity and allow bacteriophagelambda to lysogenize. J. Bacteriol. 127:1208-1216.

10. Goff, S. A., L. P. Casson, and A. L. Goldberg. 1984. The heatshock regulatory gene, htpR, influences rates of protein degra-dation and expression of the lon gene in Escherichia coli. Proc.Natl. Acad. Sci. USA 81:6647-6651.

11. Goldberg, A. L. 1972. Degradation of abnormal proteins inEscherichia coli. Proc. Natl. Acad. Sci. USA 69:422-426.

12. Goldberg, A. L., K. H. Sreedhara Swamy, C. H. Chung, andF. S. Larimore. 1983. Proteases of Escherichia coli. MethodsEnzymol. 80:680-702.

13. Goldberg, A. L., and A. C. St. John. 1976. Intracellular proteindegradation in mammalian and bacterial cells. Annu. Rev.Biochem. 45:747-803.

14. Gottesman, S., M. E. Gottesman, J. E. Shaw, and M. L. Pearson.1981. Protein degradation in E. coli: the Ion mutation andbacteriophage lambda N and cII protein stability. Cell24:225-233.

15. Gottesman, S., P. Trisler, and A. Torres-Cabassa. 1985. Regu-lation of capsular polysaccharide synthesis in Escherichia coliK-12: characterization of three regulatory genes. J. Bacteriol.162:1111-1119.

16. Gottesman, S., and D. Zipser.- 1978. Deg phenotype of Esche-richia coli lon mutants. J. Bacteriol. 113*844-851.

17. Gulletta, E., A. Das, and S. Adhya. 1983. The pleiotropic ts15mutation of E. coli is an IS1 insertion in the rho structural gene.Genetics 105:265-280.

18. Howard-Flanders, P., E. Simson, and L. Theriot. 1964. A locusthat controls filament formation and sensitivity to radiation inEscherichia coli K12. Genetics 49:237-246.

19. Huisntan, O., R. D'Ari, and S. Gottesman. 1984. Cell divisioncontrol in Escherichia coli: specific induction of the SOSfunction SfiA protein is sufficient to block septation. Proc. Natl.Acad. Sci. USA 81:4490-4494.

20. Laemmli, U. K. 1970. Cleavage of structural proteins duringassembly of the head of bacteriophage T4. Nature (London)227:680-685.

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