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JOURNAL oF BACTERIOLOGY, Mar. 1983, p. 1368-1378 Vol. 153, No. 30021-9193/83/031368-11$02.00/0Copyright 0 1983, American Society for Microbiology
Isolation and Characterization of Tn5 Insertion Mutations inthe lexA Gene of Escherichia coli
JUDY HEILIG KRUEGER, STEPHEN J. ELLEDGE, AND GRAHAM C. WALKER*Biology Department, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
Received 4 October 1982/Accepted 28 December 1982
A Mu d(Ap lac)-generated fusion of lacZ to dinD, a gene induced by DNAdamage, was used to isolate TnS insertion mutations that affect the regulation ofthe SOS responses. Three mutants were obtained that contained TnS insertionsgenetically linked to the lexA gene and had properties that suggested the mutantswere deficient in lexA expression. The lexA protein has been shown to function asthe repressor for genes involved in the SOS responses. By Southern blottingexperiments, the three TnS insertions were physically mapped to distinct loca-tions within the coding region of the lexA gene. The introduction of thesemutations in six strains carrying lacZ fusions to different damage-inducible genesresulted in high expression of 0-galactosidase in all but one of the strains. In thedinF fusion strain, lacZ expression was reduced below that seen in a lexA+background. Physical mapping studies of the dinF locus gave results consistentwith the notion that dinF is part of the lexA transcription unit and that a lexA::Tn5mutation has a polar effect on dinF expression. With certain din-lac fusion strains,a correlation was seen between the amount of P-galactosidase production and thelocation of the particular TnS insertion within the lexA gene.
Treatment ofEscherichia coli cells with any ofa variety of agents that either damage DNA orinterfere with DNA replication results in thecomplex "SOS" response, in which a set ofcellular genes are expressed at higher levels (16,21, 41). Included among these genes are umuCand umuD (induced mutagenesis) (3; S. J. El-ledge and G. C. Walter, J. Mol. Biol., in press),sulA (formerly sfiA; filamentous growth) (13),uvrA and uvrB (excision repair) (11, 17, 34, 35),himA (site-specific recombination) (25), and sev-eral din (damage-inducible) genes whose func-tion is not yet known (16). Plasmid-coded genes,such as the muc genes of pKM101 (Elledge andWalker, unpublished data) and the colicin Elgene on colEl (39), are also induced as part ofthe SOS response when these plasmids are pre-sent in a cell. All these genes are members of aregulatory network controlled by the products ofthe recA and lecA loci, which are themselvesinducible (5, 20, 23).The basic elements of the regulatory circuitry
for the genes involved in the SOS response havebeen deduced from genetic and biochemicalexperiments and were recently reviewed (21).The lexA protein appears to function as a repres-sor of all of the din genes that have beenidentified to date (3, 5, 6, 15-17, 21). Damage tocellular DNA or interference with its replicationinitiates a series of events that lead to activationof the protease activity of the recA protein (19,
33). In vitro experiments suggest that single-stranded DNA may be involved in this activa-tion (10). The lexA protein is then proteolyticallycleaved by the recA protease, and, as the poolsof lexA protein decrease, the various din genesbegin to be expressed at increased levels (15,21). The lexA binding sites for several of thesegenes were recently sequenced (21).The lexA gene was originally defined geneti-
cally by a class of dominant mutations, such asthe lexA3 allele, that blocked expression of theSOS responses and made the cells UV sensi-tive (28). These mutations are now termedlexA(Ind-) and have been shown to result in theproduction of lexA proteins that are resistant tocleavage by the recA protein (22). Two differentclasses of recessive lexA alleles were then isolat-ed, termed lexA(Ts) (formerly called tsl [29]) andlexA(Def) (formerly called spr [27]). These al-leles resulted in at least some of the SOS re-sponses being expressed either at high tempera-ture, with lexA(Ts), or at both high and lowtemperatures, with lexA(Def). The class of al-leles termed lexA(Def) seemed to result from theloss of at least some lexA function, since ambermutations mapping in the lexA gene fell into thelexA(Def) class (31).
Recently, we and others have used the Mud(Ap lac) bacteriophage (8) to generate lac oper-on fusions to various din genes, and theseproved to be useful in carrying out both genetic
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TnS INSERTIONS IN lexA 1369
(15, 21) and biochemical (15) analyses of theregulation of genes involved in the SOS respons-es. We are now using the din-lac fusions to aid inthe convenient isolation of new mutations affect-ing din gene expression. As a first step in thisanalysis, we isolated TnS insertion mutationsaffecting the lexA gene, in part because theygave alleles of this important regulatory genethat were easy to manipulate genetically, butmore importantly because they allowed us toaddress the question of whether the lexA prod-uct coded for any function essential to cellgrowth. In the course of this work, we also madesome interesting observations that suggestedthat the dinF locus lies within an operon that istranscribed from the lexA promoter.
MATERIALS AND METHODSStrain constructions. The bacterial strains used in
this study are listed in Table 1. Strains containing theplasmids pGW600 (17a) and pSE152 (Elledge andWalker, in press) were prepared by transformationof competent cells (17a). Derivatives containingthe 1exA3(Ind-) allele (from strain DM49 [28]) orlexASJ(Def) allele (from strain DM1187 [27]) wereconstructed by P1 transduction, using alleles of thenearby malE locus for selection (malE::TnS in strainRB800, obtained from R. Brent, Harvard University;malE::TnJO from strain D4703, obtained from D. Bot-stein, MIT). P1 transductions were performed basical-ly as described by Miller (26). When transducing cellsto kanamycin resistance (Kmr), we plated the mixtureof cells and phage on M9 minimal glucose platescontaining kanamycin directly after infection. Fortransductions to tetracycline resistance (Tcr), the in-fected cells were plated directly on LB plates contain-ing tetracycline. Cells containing random insertions ofTn5 were isolated as described by Krueger and Walker(17a).Media. LB and M9 minimal media for liquid and
plate cultures and X-YM broth were used as describedpreviously (17a). When desired, 5-bromo-4-chloro-3-indolyl-p-D-galactopyranoside dissolved in N,N'-di-methyl formamide was added after autoclaving to givea final concentration of 40 ,ug/ml. Drugs were addedafter autoclaving at final concentrations as follows:ampicillin, 25 ,ug/ml; tetracycline, 20 ,ug/ml; spectino-mycin, 50 p.g/ml; and kanamycin, 25 to 30 ,ug/ml.Growth of cells and ,-galactosidase assays. Cells
were inoculated and grown in M9 minimal mediumsupplemented with 0.4% glucose, vitamin B1, and therequired amino acids with vigorous shaking for 3 to 5 hto ensure that cells were in the exponential phase.Cells (5 ml) were irradiated with UV light (15 J/m2; 1.5J/m2 for uvrA strains) in 100-mm glass petri dishes (or1.6 ml of cells in 60-mm dishes). During the samplingtime, the cultures were occasionally diluted with medi-um to maintain an optical density at 600 nm of 0.2 to0.5. Duplicate samples were assayed for P-galacto-sidase as described by Miller (26), with the modifica-tion that the optical density at 600 nm was measuredon a sample diluted into an equal volume of Fixsolution (1:200 dilution of 37% formaldehyde solutionin water).
Induction of recA protein. Bacterial cultures were
grown to a density of 2 x 108 cells per ml at 30°C in M9minimal glucose medium supplemented with B1 andrequired amino acids. A 4-ml amount of each culturewas then UV irradiated in a 100-mm glass petri dishwith 100 J/m2. After 30 min, the cells were pulse-labeled for 5 min with [35S]methionine (AmershamCorp.) (5 ,uCi/ml of cells), chased for 1 min with coldmethionine (1 mg/ml final concentration), and harvest-ed. Pelleted cells were resuspended, and one-fifth ofthe sample was separated on a 12% sodium dodecylsulfate-polyacrylamide gel by the method of Laemmli(18).
Isolation of DNA. Isolation of chromosomal DNAwas done as described by Elledge and Walker (inpress). Plasmid DNA was isolated by the procedure ofTimmis et al. (40), with the modifications of Taylorand Cohen (38). The EcoRI-HindIII DNA fragmentthat contained the lexA gene was isolated by electro-elution from a gel band cut from a 0.8% agarose gel.This fragment was labeled with 32P by nick translation(32) with the modifications of Chung et al. (9). LabeledDNA was denatured by boiling for 10 min and rapidlycooled in an ice-water bath. It was separated from theresidual labeled nucleotides by ethanol precipitation.
Electrophoresis of DNA, Southern blotting, and hy-bridizations. Chromosomal DNA was digested andfragments were separated on agarose gels by theprocedures of Elledge and Walker (in press). TheDNA fragments were then transferred onto nitrocellu-lose (Schleicher & Schuell Co., no. BA85) by themethod of Southern (37). Hybridization of the labeledprobe DNA to the DNA filters was performed asdescribed by Elledge and Walker (in press).
RESULTS
Isolation of TnS insertions in lexA. To isolateTn5 insertion mutations in the lexA gene, webegan with strain GW2705, a lilac strain thatcontains both a Mu d(Ap lac)-generated fusionof lacZ to the dinD promoter/regulatory region(15, 16) and also the lexA3(Ind-) mutation (28).Since the repressor coded by the lexA3 gene isresistant to cleavage by the recA protease (19),dinD and other genes in the SOS regulatorycircuit are expressed only at a low basal level ina lexA3 background and are uninducible byDNA-damaging agents (16). This strain also car-ries a plasmid, pGW600, that overproduces tem-perature-resistant Mu repressor and thus lowersthe levels of spontaneous transposition and dele-tion formation by Mu d(Ap lac) (17a). Finally,the strain carried the sulAll (formerly sfiAll)mutation, to prevent lethal filamentation fromoccurring if a lexA(Def) (spr-like) allele of lexAwere generated (27). Derivatives of strainGW2705 containing random insertions of TnS(17a) were plated on minimal glucose mediumcontaining 5-bromo-4-chloro-3-indolyl-p-D-ga-lactoside, an indicator of 3-galactosidase activi-ty. Out of approximately 23,000 colonies, wefound 25 that expressed P-galactosidase at ele-vated levels, as indicated by the dark blue colorof the colonies on indicator-containing plates.
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1370 KRUEGER, ELLEDGE, AND WALKER
TABLE 1. Bacterial strains'Strain
GW1000GW1040GW2701GW2705GW2706GW2707GW2708GW1043
GW2709GW2710GW2711GW2712
GW1103GW2713GW2714GW2715
GW1010GW2716GW2717GW2718GW1013
GW1030GW2719GW2720GW2721
GW1060GW2723GW2724GW1063
GW1070GW2728GW2725GW2729
Genotype
recA441 sulA1l AlacUI69 thr-l leu-6 his-4 argE3 ilv(Ts) gaIK2 rpsL31As for GW1000, but dinDI::Mu d(Ap lac)As for GW1000, but dinDl::Mu d(Ap lac)/pGW600As for GW1000, but dinDl::Mu d(Ap lac) lexA3(Ind-)/pGW600As for GW1000, but dinDl::Mu d(Ap lac) lexA72::Tn5/pGW600As for GW1000, but dinDl::Mu d(Ap lac) lexA71::TnS/pGW600As for GW1000, but dinDl::Mu d(Ap lac) lexA73::TnS/pGW600As for GW1000, but dinDl::Mu d(Ap lac) lexASl(Def)
As for GW1000, but dinDl::Mu d(Ap lac) lexA72::TnS/pSE152As for GW1000, but dinDl::Mu d(Ap lac) lexA71::Tn5/pSE152As for GW1000, but dinDl::Mu d(Ap lac) lexA73::TnS/pSE152As for GW1000, but dinDI::Mu d(Ap lac) lexA51(Def)/pSE152
As for GW1000, but umuC121::Mu d(Ap lac) uvrA6As for GW1000, but umuC121::Mu d(Ap lac) uvrA6 lexA72::TnS/pGW600As for GW1000, but umuC121::Mu d(Ap lac) uvrA6 lexA71::TnSAs for GW1000, but umuC121::Mu d(Ap lac) uvrA6 lexA73::TnS
As for GW1000, but dinAI::Mu d(Ap lac)As for GW1000, but dinAl::Mu d(Ap lac) lexA72::TnSAs for GW1000, but dinAl::Mu d(Ap lac) lexA71::TnSAs for GW1000, but dinAl::Mu d(Ap lac) lexA73::TnSAs for GW1000, but dinAI::Mu d(Ap lac) lexASl(Def)
As for GW1000, but dinBI::Mu d(Ap lac)As for GW1000, but dinBl::Mu d(Ap lac) lexA72::TnSAs for GW1000, but dinBl::Mu d(Ap lac) lexA71::TnSAs for GW1000, but dinBl::Mu d(Ap lac) lexA73::TnS
As for GW1000, but uvrA215::Mu d(Ap lac)As for GW1000, but uvrA21S::Mu d(Ap lac) lexA7I::TnSAs for GW1000, but uvrA215::Mu d(Ap lac) lexA73::TnSAs for GW1000, but uvrA215::Mu d(Ap lac) lexA51(Def)
As for GW1000, but dinFl::Mu d(Ap lac)As for GW1000, but dinFl::Mu d(Ap lac) lexA3(Ind-) maIE::TnlOAs for GW1000, but dinFl::Mu d(Ap lac) lexA71::TnSAs for GW1000, but dinFl::Mu d(Ap lac) lexA51(Def) malE::Tn5
I The nomenclature used is that of Bachmann and Low (2) and Campbell et al. (7).
Since strains containing the IexA3 allele aremore sensitive to killing by UV irradiation thanare strains containing either a IexA+ or alexA(Def) allele, we tested those 25 derivativesfor their sensitivity to killing by UV. Threestrains, GW2706, GW2707, and GW2708, werefound to be much more resistant to killing thanwas their parent and were therefore candidatesfor strains carrying insertions of TnS in the lexAgene.The level of P-galactosidase expression in
these three strains was examined more carefullyby assaying for activity in growing cultures withand without exposure to UV radiation. Figure 1shows the levels of ,3-galactosidase expressionin dinDI::Mu d(Ap lac) strains carrying thesethree TnS insertions compared with the lev-els in derivatives carrying lexA+, lexA3, or
lexASl(Def) (formerly spr-SJ) alleles. The TnS-containing strains resembled the lexASi strain inhaving high levels of 0-galactosidase that werenot appreciably affected by treatment with UVradiation. In fact, the expression of P-galacto-sidase in two of the dinDl ::Mu d(Ap lac) strainscarrying TnS insertions was even higher than inthe lexASI-containing strain. These observa-tions were consistent with strains GW2706,GW2707, and GW2708 carrying lexA(Def) allelesof the lexA gene, but did not establish that theputative lexA(Def) mutations were caused by theTnS insertions.
Genetic analysis of the klxA::TnS mutations.We next checked that the TnS insertions in thesestrains were genetically linked to the mutationscausing their elevated ,-galactosidase expres-sion. P1 lysates grown on these strains were
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TnS INSERTIONS IN lexA 1371
nomenon is caused by Tn5 sometimes integrat-ing by transposition rather than by homologousrecombination during the transduction proce-
500 - dure.We then tested whether the TnS insertions in
lexA71 ::Tn5 the three strains GW2706, GW2707, andGW2708 were genetically linked to the lexAlocus. P1 lysates grown on these three strainswere used to transduce two independent
o 400 dinFI::Mu d(Ap lac) strains to Kmr; the Muox lexA 72 Tn5 d(Ap lac) insertions in dinF are tightly linked to
o lexA by P1 transduction (16). The frequency ofampicillin-sensitive derivatives (indicating the
C loss of Mu d[Ap lac]) among the Kmr transduc-10. tants was 88% (298 of 339), 92% (183 of 200), and. 300 69% (186 of 270) for P1 lysates grown on strains:Z GW2706, GW2707, and GW2708, respectively.U Another indication that the TnS insertions inX~ . ~8lexA5I (Def) these strains had inserted in the lexA gene was
o the result of an experiment in which we intro-duced plasmid pSE152 (Elledge and Walker, in
200 press), a spectinomycin-resistant derivative ofpRB160 (5) that produces high levels of the lexA
lexA 73::Tn5 protein, into strains GW2706, GW2707, and< o GW2708. Introduction of this plasmid into a
lexA+ strain makes the strain UV sensi-tive, mimicking the noninducible phenotype of
100 lexA3(Ind-) mutants. We expected that if thelexA + TnS insertions were within the lexA gene, then
the introduction of pSE152 would result in UVsensitivity and reduced expression of ,B-galacto-sidase. We transformed strains GW2706,
lexA3 (I nd-) GW2707, and GW2708 and a control strain,L -3 i GW1043, that contains dinDi::Mu d(Ap lac) and
1 2 3 4 lexA51(Def) with pSElS2, selecting for spectino-t Time (hr) mycin resistance. All four strains became sensi-
FIG. 1. Time course of ,3-galactosidase expression tive to UV irradiation after acquiring pSE152.in regulatory mutants of the dinDl::Mu d(Ap lac) Furthermore, the levels of ,-galactosidase werestrain after UV irradiation. Samples were periodically reduced to that observed in a dinDi ::Mu d(Aptaken from log-phase cultures grown at 30°C in M9- lac) lexA3 strain and were not inducible by UVglucose medium supplemented with thiamine and re- (Table 2).quired amino acids. At the time indicated by the Other phenotypes of lexA::TnS strains. Strainsarrow, half of the culture was irradiated with UV (15 J/ GW2706, GW2707, and GW2708 had two othermi). The lag seen previously in ,-galactosidase induc- characteristics similar to lexASi strains. First,tion in a dinDl::Mu d(Ap lac) fusion strain (16) was chesthre strai to cA51 the rst,not seen when the UV dose was lowered from 60 to 15 since these three strains also carry the recA44JJ/m2 (C. J. Kenyon, Ph.D. thesis, K. H. Paek, person- (formerly tif-J) mutation, it was possible to seeal communication). Symbols: 0, no UV irradiation; 0, whether they resembled lexA51 recA441 (tif-1)after UV irradiation. strains in giving clear plaques with cI+ X bacte-
riophage (27). Two of these strains, GW2706 andused to transduce the parent strain GW2705 GW2707, did give relatively clear plaques with(dinDI::Mu d[Ap lac] lexA3) to kanamycin cI+ X at 30°C on X plates. With strain GW2708resistance (Kmr). The Kmr transductants were the plaques were only slightly clearer than withfound to express high levels of 3-galactosidase lexA+ strains.at frequencies of 66 to 100%. We therefore Second, all three strains constitutively pro-concluded that for each of the three strains the duce high levels of recA protein as assayed byTnS insertion was linked to the regulatory muta- autoradiography of [35S]methionine-labeled pro-tion, since we usually see cotransduction fre- teins separated by sodium dodecyl sulfate-poly-quencies of less than 100% when TnS-generated acrylamide gel electrophoresis. As expected,insertion mutations are transduced and kanamy- with the uninduced parent strain we did not see acin resistance is selected. Presumably, this phe- 40-kilodalton band either by Coomassie blue
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1372 KRUEGER, ELLEDGE, AND WALKER
TABLE 2. 1-Galactosidase expression in lexA::TnS derivatives of the dinDl::Mu d(Ap lac) fusion straincontaining pSE152 (lexA+)
,B-Galactosidase activityaStrain Relevant genotype Without UV With UV
GW2701 dinDl::Mu d(Ap lac) 23 142GW1043 dinDI::Mu d(Ap lac) lexASl(Def) 249 230GW2706 dinDI::Mu d(Ap lac) lexA72::TnS 404 370GW2707 dinDI::Mu d(Ap lac) lexA7l::Tn5 417 440GW2708 dinDI::Mu d(Ap lac) lexA73::Tn5 157 148GW2705 dinDl::Mu d(Ap lac) lexA3 12 12GW2712 dinDI::Mu d(Ap lac) lexASl(Def)/pSE152 12 14GW2709 dinDI::Mu d(Ap lac) lexA72::TnS/pSE152 11 11GW2710 dinDl::Mu d(Ap lac) lexA7l::Tn:5/pSE152 12 13GW2711 dinDI::Mu d(Ap lac) lexA73::TnS/pSE152 11 12
a Early-log-phase cultures, grown at 30°C in M9-glucose medium supplemented with the required amino acidsand thiamine, were divided, and half ofeach was UV irradiated with 15 JIm2. After 2.5 h of additional growth, thesamples were assayed for 0-galactosidase activity. Values (units of enzyme per optical density measurement at600 nm) are the average of two determinations.
staining or by autoradiography, but such a bandwas seen after UV treatment. In contrast, withthe three Tn5 derivatives a 40-kilodalton bandwas visible in the gel stained with Coomassieblue and on an autoradiograph of the gel withoutinduction of the strains.
Physical mapping of the lexA::TnS insertions.All the evidence presented above suggested thatthe three TnS insertions had actually occurred inthe lexA gene. To prove this unambiguously, wedecided to physically map the positions of theseTnS insertions by Southern blotting experiments(37). A lexA-specific probe was prepared frompSE152 that consisted of an approximately 950-base-pair segment extending from an EcoRI siteabout 70 base pairs in front of the start site forthe lexA mRNA to an HindIII site about 250base pairs past the terminal codon of the lexAgene (5). With chromosomal DNA from theparent strain, GW2705, the lexA-specific probehybridized to only one fragment in an EcoRI-Sall or an EcoRI-BamHI double digest (Fig. 2),implying that the hybridizable fragments were atleast as large as the probe. The mobilities of thehybridizing fragments on the gel correspondedto sizes of 10 and 12 kilobases (kb), respectively,which agreed with previously reported mappingdata (24).With the three Tn5 insertion strains, two
fragments hybridizing to the lexA-specific probewere seen in both of the double digests, implyingthat new restriction sites had been introducedwithin the region hybridizing to the probe andthus that the TnS insertions had occurred withinthe 950 base pairs defined by the probe. There isonly one BamHI and one SaI site in Th5 (14). Inone of the TnS insertion strains, GW2708, therelative sizes of the small bands in the two
1 2 3 4 b 6 7 8
1 0 kb -
FIG. 2. Location of Tn5 insertions in the lexAgene. Chromosomal DNA from the dinDl::Mu d(Aplac) strain and its TnS derivatives was cleaved withthe indicated restriction enzymes, fractionated on0.8% agarose gels, and blotted to nitrocellulose asdescribed in the text. The probe employed was theEcoRI-HindIII fragnent from pSE152 containing thelexA gene and adjacent E. coli DNA (see text). Lanes:1 and 2, strain GW2701 (lexA') DNA; 3 and 4,strain GW2706 (lexA72::Tn5) DNA; 5 and 6, strainGW2707 (lexA71::Tn5) DNA; 7 and 8, strain GW2708(lexA73::TnS) DNA. The DNAs in lanes 1, 3, 5, and 7were digested with EcoRI and Sall. The DNAs inlanes 2, 4, 6, and 8 were digested with EcoRI andBamHI.
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TnS INSERTIONS IN lexA 1373
Soil SmHI HincII419 .4 Tn5
BumHI EcoRI1
(X)(i)I -11
I lexA I
FIG. 3. The upper line is a partial restriction map of TnS, showing the unique Sall and BamHI sites and thetwo HinclI sites closest to the ends of the element (1, 14). TnS is not cleaved by EcoRI. The second line is a
partial restriction map of the lexA region of E. coli, indicating the EcoRI and BamHI sites. This is in agreementwith the restriction map of Miki et al. (24). The heavy line indicates the DNA carried by the probe; this region isexpanded in the bottom line, indicating the position of the HinclI restriction sites and the extent of the lexA gene
(12). The locations of the three Tn5 insertions in the lexA gene are also shown. Symbols: +, TnS has inserted inthe orientation presented in the top line; -, TnS has inserted in the opposite orientation.
double digests were reversed compared withthose in strains GW2706 and GW2707, indicatingthat insertion of the TnS element in strainGW2708 had occurred in the orientation oppo-site those in the other two strains.Using a variety of restriction endonuclease
digestions, we mapped the positions of the TnSinsertions in strains GW2707, GW2706, andGW2708 to a HincII fragment entirely within thegene and lying 30 (±20), 280 (±40), and 390(±30) base pairs, respectively, downstreamfrom the initial codon for the lexA protein (Fig.3). The lexA alleles in these strains were giventhe following designations: lexA71::TnS (strainGW2707), lexA72: :TnS (strain GW2706), andlexA73::TnS (strain GW2708). It is interesting tonote that the dinDI::Mu d(Ap lac) strain inwhich the TnS insertion occurred closest to the5' end of the lexA gene (1exA71::TnS) had thehighest level of 3-galactosidase expression,and the strain in which the TnS insertion oc-curred closest to the 3' end of the lexA gene(lexA73::TnS) had the lowest level of P-galacto-sidase expression. In the latter strain, a polypep-tide that was 60 to 70% of the size of the lexAprotein could be synthesized.
Effect of lexA::TnS mutations on regulation ofother damage-inducible genes. Since the Tn5insertions we isolated were within the lexA gene,cells carrying these mutations could not makethe lexA protein and would have the phenotypecaused by a lexA null mutation. Thus, we ex-
pected that introduction of the Tn5 insertionsinto strains containing Mu d(Ap lac) fusions tothe promoters of other damage-inducible geneswould result in the production of high levels ofP-galactosidase in those strains. We introducedthe lexA::TnS insertions into strains containing
Mu d(Ap lac)-generated operon fusions of lacZto dinA, dinB, umuC, uvrA, and dinF. For thefirst four strains, the prediction was borne out-the level of P-galactosidase in these strains wasvery high (Table 3). Interestingly, in contrast tothe dinD fusion strain, for the dinA, dinB, anduvrA fusion strains introduction of the differentlexA::TnS alleles and the lexASl(Def) mutationdid not cause different levels of 3-galactosidaseexpression. In the umuCJ21::Mu d(Ap lac)strains, the P-galactosidase level in the mutantcontaining the lexA71::Tn5 allele was about 40%higher than in strains containing either of theother lexA::Tn5 mutations.An unexpected result was obtained when the
lexA71 :TnS mutation was introduced into a
dinFl::Mu d(Ap lac) IexA3 strain. The level ofP-galactosidase seen in this derivative was nothigher, but rather lower than that of the IexA3parent strain. This was initially surprising, sincewe had previously shown that the introductionof a lexASI mutation into a dinFi ::Mu d(Ap lac)strain leads to high levels of 3-galactosidaseexpression (15). We considered two possibleexplanations for these low levels of 3-galacto-sidase expression. (i) The TnS element mighthave transposed to new locations during thestrain construction, or (ii) the TnS element,which is polar (4), was interfering with theexpression of the dinF fusion.The first possibility did not seem likely, since
the lexA7J::TnS mutation was transduced fromthe dinFI::Mu d(Ap lac) strain into a lexA3background, and the Kmr transductants becameUV resistant and constitutively expressed therecA protein at high levels, implying that thecells had indeed acquired a lexA(Def) mutation.Furthermore, the Tn5 insertion in this strain was
I kb
HinC.4
EcoRI
I H _
I I
I HincI
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1374 KRUEGER, ELLEDGE, AND WALKER
TABLE 3. P-Galactosidase expression in din-lac strains containing lexA::TnS(Def) alleles
Sn-Galactosidase activityaStrain Relevant genotype WtotU ihUWithout UV With UV
GW1103 umuC121::Mu d(Ap lac) uvrA6 14 75GW2714 umuC121::Mu d(Ap lac) uvrA6 lexA71::TnS 286 279GW2713 umuC121::Mu d(Ap lac) uvrA6 lexA72::TnS 203 208GW2715 umuC121::Mu d(Ap lac) uvrA6 lexA73::TnS 214 210
GW1010 dinAl::Mu d(Ap lac) 14 50GW2717 dinAI::Mu d(Ap lac) lexA7J::TnS 102 101GW2716 dinAI::Mu d(Ap lac) lexA72::TnS 108 104GW2718 dinAl::Mu d(Ap lac) lexA73;:TnS 101 100GW1013 dinAI::Mu d(Ap lac) lexASl(Def) 115 114
GW1030 dinBI::Mu d(Ap lac) 24 107GW2720 dinBl::Mu d(Ap lac) lexA71::TnS 294 297GW2719 dinBl::Mu d(Ap lac) lexA72::TnS 300 260GW2721 dinBl::Mu d(Ap lac) lexA73::TnS 290 314
GW1060 uvrA2I5::Mu d(Ap lac) 123 416GW2723 uvrA21S::Mu d(Ap lac) lexA7I::TnS 882 809GW2724 uvrA215::Mu d(Ap lac) lexA73::TnS 828 803GW1063 uvrA215::Mu d(Ap lac) lexASI(Def) 910 895
GW1070 dinFl::Mu d(Ap lac) 60 218GW2728 dinFl::Mu d(Ap lac) IexA3(Ind-) 26 23GW2725 dinFl::Mu d(Ap lac) lexA71::TnS 5 5GW2729 dinFl::Mu d(Ap lac) lexASl(Def) 313 306
a After growth at 30°C on M9-glucose medium supplemented with the required amino acids and thiamine,cultures were divided, and half of each was UV irradiated with 15 JMm2. After 2.5 h of additional growth, thesamples were assayed for j-galactosidase activity. Values (units of enzyme per optical density measurement at600 nm) are the average of two determinations.
linked to malE by P1 transduction, as is lexA,and the results of Southern blotting experimentswere consistent with this strain containing a TnSinsertion in the lexA gene. In addition, threeother independent transductants of lexA71::TnSinto the dinFI::Mu d(Ap lac) strain had prop-erties similar to the dinF)::Mu d(Ap lac)lexA71::Tn5 strain (GW2725) that we character-ized in detail.The second possibility thus seemed more like-
ly, especially in view of the fact that thedinFI::Mu d(Ap lac) insertion maps very closeto lexA (16). This close linkage of dinF to thelexA locus was confirmed during our construc-tion of the dinF::Mu d(Ap lac) lexA71::TnSderivative. For example, in one screen of 300Kmr transductants, only 35 were still Apr (stillcarried the Mu d[Ap lac] bacteriophage), andonly one of the 35 had lost the UV sensitivity ofthe original lexA3 parent. The other 34 transduc-tants were presumed to have resulted fromtranspositions of TnS during the transduction.Initial mapping of the dinFI::Mu d(Ap lac) inser-tion by the Southern blotting technique suggest-ed that it was in fact quite close to lexA (C. J.Kenyon, Ph.D. thesis, Massachusetts Institute
of Technology, Cambridge, 1981), and we decid-ed to map the insertion more carefully.
Physical mapping of the dinFl::Mu d(Ap lac)insertion. To map the position of the dinFl::Mud(Ap lac) mutation in strain GW1070 (16), weused the same lexA-specific probe describedabove. The sizes of the fragments obtained inthree restriction digestions of the parental (strainGW1000) DNA (Fig. 4) agreed with previousmapping of the area by other investigators (5, 12,24). With the same digestion conditions, differ-ently sized fragments were obtained with DNAfrom the dinF: :Mu d(Ap lac) derivative ofstrain GW1000. This can be seen most easilywith the EcoRI single and EcoRl-PstI doubledigestions (Fig. 4); the BamHI fragments werequite similar in size by coincidence of the site ofintegration and location of restriction sites. Thedifference seen with the EcoRI-PstI double di-gestion indicated that the Mu d(Ap lac) insertedbetween the EcoRI and PstI sites (Fig. 5). Inaddition, EcoRI-KpnI double digestions of theparentai strain and of the dinFi::Mu d(Ap lac)strain showed that the insertion occurred be-tween the EcoRI and KpnI sites (data notshown). The fragment sizes placed the insertion
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TnS INSERTIONS IN lexA 1375
*12 kb--10
'-6.5
FIG. 4. Mapping of the Mu d(Ap lac) insertion atthe dinF locus. Chromosomal DNA from thedinFl::Mu d(Ap lac) strain and its parent strain wascleaved with the indicated restriction enzymes, frac-tionated on 0.8% agarose gels, and blotted to nitrocel-lulose as described in the text. The probe employedwas the EcoRI-HindIII fragment of pSE152 containingthe lexA gene and adjacent E. coli DNA. Lanes: 1, 2,and 3, strain GW1000 DNA; 4, 5, and 6, strainGW1070 DNA. The DNAs in lanes 1 and 4 weredigested with EcoRI, in lanes 2 and 5 with EcoRI andPstl, and in lanes 3 and 6 with BamHI.
at approximately 1.4 ± 0.2 kb from thie 3' end ofthe lexA coding region.The location of the EcoRI and PstI sites
nearest the ends of the Mu d(Ap lac) phage and
EcoRI Bon,Hl Psf EcoRI BomHt BamHI1, J, 1L %L J
/5Cb5kb,I
somHIEcoRI
9KpnI PstlI
J~ %LI
the location and direction of transcription of thelacZ gene are shown in Fig. 5 (M. O'Conner,personal communication). The fact that the frag-ment in the EcoRI-PstI double digest of thedinFi insertion was not smaller than the frag-ment in the single EcoRI digest of this strainindicated that the Mu d(Ap lac) insertion at thedinF locus occurred in the orientation shown inFig. 5. Thus, the lacZ gene of dinFI::Mu d(Aplac) is not only quite close to the lexA gene but isalso transcribed in the same direction. We foundno evidence for rearrangements of the chromo-somal DNA in this analysis or in any of theSouthern hybridizations we carried out, includ-ing a comparison of dinF1::Mu d(Ap lac) strain(GW1070) and its lexA71::Tn5 derivative(GW2725).These observations are consistent with a mod-
el in which the dinF locus is in the same operonas the lexA gene but downstream. The low levelof 3-galactosidase expression seen in dinF1::Mud(Ap lac) derivatives containing the lexA7J :TnSmutation but not the lexASJ(Def) mutation mightthen be due to a polar effect of the Tn5 insertionin the lexA gene on transcription at the dinFlocus.
DISCUSSIONUsing a strain containing an operon fusion of
lacZ to the dinD promoter/regulatory region, weisolated Tn5-induced mutations in the lexA geneby screening for cells that produced elevatedlevels of P-galactosidase in the absence of aninducing signal. Since we previously presentedevidence that lexA functions as the direct repres-sor of expression from the dinD promoter (15),this class of regulatory mutations was expected.
EcoRI PstI'I- 4 Mu d(Ap /oc)
BomHI EcoRI%i 1b
dinF/::Mu d(Ap /c)I-1
lexA IFIG. 5. The upper line is a partial restriction map of Mu d(Ap lac), indicating the EcoRI, BamHI, and PstI
restriction sites and the location and orientation of the lac genes (M. O'Conner, personal communication). Thesecond line indicates the BamHI and EcoRI sites in the lexA region and the KpnI and PstI sites closest to the 3'end of lexA. These are in agreement with the data of Miki et al. (24). The EcoRI-KpnI fragment is expanded in thebottom line, indicating the location and direction of transcription of the lexA gene (12) and of the Mu d(Ap lac)insertion in dinF.
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1376 KRUEGER, ELLEDGE, AND WALKER
Furthermore, by physically mapping the TnSinsertions we showed that the regulatory muta-tions were actually caused by insertions of TnSin the lexA gene rather than by Tn5-generateddeletions or by point mutations that were geneti-cally linked to the TnS insertions. The strategyof using Mu d(Ap lac) to conveniently generatelac operon fusions to genes of interest and thenusing TnS insertion mutagenesis to isolate cer-tain classes of regulatory mutations has provedto be a useful one and results in regulatorymutations that are easy to manipulate genetical-ly (17a, 30).The lexA71::TnS insertion in strain GW2707
maps at approximately the 30th (±25) base pairof the coding region of the lexA gene and thusprevents synthesis of almost all of the lexAprotein. Therefore, in strains carrying a sulAmutation (to prevent continuous filamentousgrowth and consequently cell death), the lexAprotein is not required for any function essentialfor cell growth, although such strains carryingthe lexA71::Tn5 allele do grow somewhat slow-ly. Amber mutants of lexA have been reportedpreviously (31), but it was not possible to con-clude unambiguously that the lexA gene productwas nonessential, since the positions of theamber mutations within the lexA coding regionwere unknown. In addition, our results suggestthat any functions coded for downstream of lexAthat are transcribed from only the lexA promoterare also unnecessary under these conditions,since the lexA71::TnS insertion is apparentlystrongly polar on the expression of downstreamgenes. Finally, since only a small fragment of thelexA protein could be made in a lexA71::TnSstrain, all genes that are negatively regulatedsolely by the lexA protein should be completelyderepressed, including the truncated lexA gene.On the other hand, the lexA73::TnS insertion
in strain GW2708 occurred at a position in thelexA coding sequence that could allow 60 to 70%oof the lexA protein to be translated. This proteinfiagment might have residual binding affinity forits operator sites and could explain the differ-ence seen in 3-galactosidase expression betweendinDl::Mu d(Ap lac) derivatives containing ei-ther this insertion or lexA71::TnS. This suggeststhat the amino-terminal half of the lexA proteincodes for the DNA binding site in a mannersimilar to the X repressor (36). This explanationis consistent with the work of two groups whocloned fragments of lexA that have lost thesequences coding for about 10 to 25% of thecarboxy end of the lexA protein. With theseplasmids, they reported complementation ofsome of the phenotypes of lexA(DefT) mutants(12, 24). Since there are some indications thatthe lexA protein exists as a dimer (6, 12, 21), thereduced efficiency of repression by the
lexA73::TnS gene product could possibly resultfrom a decrease in the ability of the protein toform dimers.
In strains carrying the lexA72::Tn5 mutation,a lexA fragment of approximately 40 to 50%o ofthe wild-type length could be made. This isabout the probable length of the amino-terminalfragment of recA-cleaved lexA protein (22). Theslightly reduced expression of dinD in alexA72::TnS mutant as opposed to that in alexA71::TnS mutant could result from residualbinding of the truncated lexA product to the lexAbinding site of the dinD gene.
If we are correct in our interpretation of theproperties of the truncated lexA polypeptidesthat could be made in the three lexA insertionmutants, then the behavior of din-lac fusions instrains carrying the various lexA::Tn5 mutationsmight give some preliminary indication of therelative strength of the lexA binding sequencefor each of these genes, and would thus repre-sent a genetic technique for predicting relativeKd's of a repressor binding to different operatorsequences. For example, the dinDI::Mu d(Aplac) fusion is more strongly derepressed in thepresence of a lexA71::Tn5 mutation than it is inthe presence of lexA72::TnS or lexA73::Tn5 mu-tations; the umuC121::Mu d(Ap lac) fusion ismore strongly derepressed in the presence oflexA71::TnS and lexA72::TnS mutations than itis in the presence of the lexA73::TnS mutation;and the dinAl::Mu d(Ap lac) fusion is equallyderepressed in the presence of any of the threelexA insertion mutations. If it is assumed thatthe strength of a lexA binding sequence is relatedto its ability to bind truncated lexA proteins,then the relative strengths of the lexA bindingsequences would be dinD > umuC > dinA.After UV irradiation, P-galactosidase levels
did not increase in dinDl::Mu d(Ap lac) strainscontaining any one of the lexA::TnS insertions(Fig. 1). This was not surprising, since theprotein fragment coded for by lexA71::TnS isvery small, should have no repressor activity,and should not contain the cleavage site for therecA protease, and the truncated protein oflexA73: :Tn5 should contain the modified se-quence of 1exA3(Ind-) protein, which is muchmore resistant to cleavage by the recA protease(19). The lexA72::TnS protein might or might notcontain the lexA3 cleavage site, but in eithercase should not be a good substrate for recAprotease.
Based on the evidence (i) that the dinFl::Mud(Ap lac) insertion mapped close to the lexAgene and is transcribed in the same direction, (ii)that a TnS insertion in the lexA gene appeared todrastically reduce expression of ,B-galactosidasein a dinFI . :Mu d(Ap lac) strain, and (iii) that thisintroduction of lexA7J::TnS into the dinFl::Mu
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VOL. 153, 1983
d(Ap lac) did not cause any detectable DNArearrangements, we presume that transcriptionfrom the lexA promoter continues as far as thedinF locus in a wild-type strain. This means thatif no function is destroyed by the dinFi::Mud(Ap lac) insertion that is required for subtleregulation of the lexA gene, then ,B-galactosidaseexpression in a dinFI::Mu d(Ap lac) strain couldbe used to monitor easily the expression of thelexA transcript and presumably of the lexA pro-tein itself.
In dinF ::Mu d(Ap lac) strains, there is suffi-cient sequence between the end of the lexA geneand the position of the dinF insertion to allowthe putative lexA transcript to code for at leastone more protein. Miki et al. (24) cloned thisregion and saw a 35,000-dalton protein synthe-sized in vitro that maps at the approximatelocation of the dinFI insertion.
ACKNOWLEDGMENTS
We acknowledge many helpful discussions with C. Kenyon,K.-H. Paek, P. Pang, K. Perry, S. Winans, and the othermembers of the laboratory during the progress of our work.We also thank L. Withers for her help in preparing themanuscript, M. O'Conner for sharing his unpublished restric-tion data with us, and D. Botstein, R. Brent, and D. Mount forproviding bacterial strains.This investigation was supported by Public Health Service
grant GM28988 from the National Institute of General MedicalSciences. S.J.E. was supported by Training Grant ES07020from the National Institutes of Health. G.C.W. was a RitaAllen Scholar.
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