JOURNAL OF BACTERIOLOGY, May 1980, p. 439-4460021-9193/80/05-0439/08$02.00/0

Vol. 142, No. 2

Polarity of Tn5 Insertion Mutations in Escherichia coliDOUGLAS E. BERG,* ALISON WEISS,t AND LYLE CROSSLAND

Department ofMicrobiology and Immunology and Department of Genetics, Washington University MedicalSchool, St. Louis, Missouri 63110

We assessed the effect of insertions of the kanamycin resistance transposonTn5 in the lac operon of Escherichia coli on the expression of distal genes lacYand lacA (melibiose fermentation at 41°C and thiogalactoside transacetylasesynthesis, respectively). Every insertion mutation tested (41 in lacZ and 23 inlacY) was strongly polar. However, approximately one-third of the insertionmutants expressed distal genes at low levels due to a promoter associated withTn5. To localize this promoter, we (i) reversed the orientation of Tn5 at severalsites and (ii) replaced wild-type Tn5 with several substitution derivatives whichlack Tn5's central region. Neither alteration changed the expression of distalgenes. Thus, in contrast to transposons IS2 and TnA, Tn5's ability to turn ondistal gene expression is not due to a promoter in its central region and thereforeis not dependent on the overall orientation of Tn5 in the operon. Our resultssuggest that the promoter is within 186 base pairs of the ends of Tn5. It is possiblethat the promoter is detected in only a fraction of insertions because it overlapsTn5-target sequence boundary.

Transposons are specialized DNA segmentswhich insert into many sites in bacterial ge-nomes. Those which encode antibiotic resistanceprovide powerful tools for microbial genetics,because their insertion into new sites generatesmutations in which the mutant allele is linkedto the selectable resistance trait (2, 14, 22; D. E.Berg and C. Egner, submitted for publication).

In most cases studied, transposon insertionmutations are strongly polar because normaltranscription in the operon distal to the trans-poson's insertion site is prevented by termina-tion signals present in the transposon. However,for insertions of two transposons, IS2 and TnA,only about half of the mutations are stronglypolar. In the remainder of IS2 and TnA inser-tions, a promoter within the transposon causesconstitutive expression of distal genes (9, 10, 14,19,20,24).The present study focuses on the polarity

exerted by the kanamycin-neomycin resistancetransposon Tn5 (3, 8) and on its ability to turnon the expression of distal genes. This elementis 5,700 base pairs long and consists of a centralunique sequence region containing the resistancegene flanked by inverted repetitions 1,500 basepairs long. It transposes at high frequency, caninsert into many sites in a gene, and has servedas a useful mutagen in numerous studies (2, 3, 4,14, 22).This study shows that insertions of Tn5 are

polar and that, although the majority of inser-t Present address: Department of Microbiology, University

of Washington, Seattle, WA 98195.

tions allow no distal gene expression, approxi-mately one-third do cause low constitutiveexpression of distal genes. It had seemed byanalogy with IS2 and TnA that the expressionof this Tn5-associated promoter might dependon the orientation ofTn5 within the operon. Ourexperiments show, however, that reversal of theorientation of Tn5 does not alter its effect ondistal gene expression. Our use of substitutionand insertion derivatives of Tn5 (see Fig. 1)suggests that the promoter is within 186 basepairs of the end of the element and may actuallyoverlap the Tn5-target sequence boundary.

MATERIALS AND METHODSPhage and bacterial strains. The Escherichia

coli K-12 bacterial and A phage strains used are listedin Tables 1 and 2. A map of Tn5 and its derivatives isshown in Fig. 1. Procedures for growing and crossingA phages and for selection and thermal induction of AcI857 lysogens (1-3), for bacterial crosses (5, 16; Bergand Egner, submitted for publication), and for trans-formation with plasmid DNA (6) have been described.lac::Tn5 insertion mutations were transduced into newgenetic backgrounds by using phage P1CM and selec-tion for Kanr Lac- transductants on lactose-tetrazo-lium-kanamycin agar.

Media. LN broth (10 g of Humko-Sheffield N-Zamine type A, 5 g of Difco yeast extract, 10 g of NaCl,and 30 mg of thymine per liter, adjusted to pH 7.3with NaOH) was used for routine growth of bacteria.N broth (identical to LN broth except for omission ofyeast extract) was used for growth and for lysogeni-zation by phage A. The minimal medium E has beendescribed (25). Solid medium was prepared with 1.5%agar (Difco) for most plates, 1.0 and 0.6% agar (Difco)

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440 BERG, WEISS, AND CROSSLAND

TABLE 1. Bacterial strainsStrain Genotype Source or Reference

DB1163 F- A(proB-lac) tip str Reference 2DB1446 HfrC proC met str D. Kennell, strain 390DB1470 F- ApE5 trpR tna Laboratory collection"DB1504 F- A(proB-lac) AtrpE5 trpR tna A(proB-lac) derivative of DB1470DB1506 F' proB' lac/lA(proB-lac) AtrpE5 trpR tna Transfer of F'proB' lac' episome from

strain CSH23 (reference 17) to DB1504DB1506-124 F'proB' lacZ124::Tn5(Kanr)/A(proB-lac) By P1 transduction from lacZl24::Tn5

AtipE5 trpR tna mutant of DB1446DB1506-204 Identical to DB506-124 except that wild-type By P1 transduction from lacP204::Tn5

Tn5 is present at the lacP204 site derivative of DB1446DB1508-124 F'proB+ lacZ204::Tn5-410(Trp)/A (proB-lac) By replacement of wild-type Tn5 with Tn5-

AtYpE5 trpR tna 410 as described in Materials and MethodsDB1512 F- A(proB-lac) AtrpE5 trpR tna str UV-induced Strr derivative of DB1504DB1544 F- A(proB-lac) ara thi str [pMC4]b M. Calos (7)

a DB1470 was constructed by P1CM transduction (16) of tpE5 (15) into a W3110 trpR tna A(trpE-A) strainfrom N. Franklin, selecting for growth on indole, which requires trpAB+ function. DB1504 was generated bytransfer of the proB-lac deletion of CSH63 (16) into DB1470.

b Plasmid pMC4 encodes Tetr, and contains an EcoRI fragment of a 480 lac phage which contains lacI, andall but the last 50 base pairs of lacZ (7). It does not complement lacZ::Tn5 insertion mutations.

TABLE 2. Phage strainsPhage Transduces Source or reference

1. A b221 c26 - Reference 22. A b221 cI857 Tn5 Kan' Reference 23. A b221 c1857 Tn5-131 Tetr Berg and Egner, submitted for publication4. A b221 cI857 Tn5410 TrpE+ Berg and Egner, submitted for publication5. A b221 cI857 Tn5-135 Tetr Recombination between phage 4 and plasmid ColEl::Tn5-135

from R. Jorgensen6. A b221 c1857 Tn5-137 Tett Recombination between phage 4 and plasmid ColEl::Tn5-137

from R. Jorgensen as described by Berg and Egner(submitted for publication)

for bottom and top agar for phage growth, respectively,and 1.5% Noble agar (Difco) for minimal lactose andmelibiose agar plates. Lactose and melibiose fermen-tation were scored on MacConkey agar (Difco) or onTTC agar (50 mg of triphenyl tetrazolium chloride, 4g of nutrient broth, 3.5 g of peptone, 3 g of yeastextract, and 15 g of agar per liter) supplemented with1% lactose or melibiose. When needed, kanamycin orneomycin sulfate was added to a final concentrationof 25 pg/mL

Isolation of lac::Tn5 insertion mutants. ac::Tn5 insertion mutants were generated by infection ofLac+ Kan! strain DB1446 with X b221 cI857 rex::Tn5,grown for 30 min at 32°C in LN broth to permitexpression of Kanr, and then grown for at least fivegenerations in LN broth containing 25p,g ofkanamycinsulfate per ml. The cultures were diluted and platedon lactose-tetrazolium-kanamycin plates to yield 500to 1,000 colonies per plate. Approximately 0.1% of theKan' colonies were Lac- (lac::Tn5). Only one mutantwas kept from each infected culture.

lacZ and lacY insertion mutants were distinguishedby the inability of lacZ to hydrolyze Xgal (5-bromo-4-chloro-3-indolyl-,B-D-galactoside). LacZ- colonies arewhite, and lacZ Y colonies are blue on media contain-ing this dye (16).Replacement of Tn5 elements. Replacement of

wild-type Tn5(Kanr) at site lacZ124 by the substitu-

tion derivative Tn5-410(Trp) is diagrammed in Fig. 2.The Trp- lacZl24::Tn5 strain DB1506-124 was lysO-genized by phage A b221 c1857 Tn5-410(Trp). The b221deletion makes this phage unable to integrate into thebacterial attachment site, and consequently stable ly-sogens are rare (2); they generally reslt from a singlecrossover between homologous portions of invertedrepetitions of wild-type Tn5 present in lacZ and theTn5-410(Trp) derivative present in phage A. Theselysogens were selected by their Trp+ A immune phe-notype at 32°C.

Nonlysogenic segregants from these lysogens wereselected by their ability to form colonies at 41°C, atemperature which induces A c1857 prophages. Suchheat-resistant nonlysogens are formed at frequenciesof 10-2 to 10-3. Approximately half are Kan' Trp+ andmust be formed as the result of a second crossover onthe other side of the inhomology between wild-typeTn5 and Tn5-410, as depicted in Fig. 2.

Replacements of wild-type Tn5 by Tn5-131(Tetr)and Tn5-132(Tetr) were generated in the same way,using ATn5-131 and ATn5-132 phages and selection forTetr instead of Trp+. Wild-type Tn5(Kan') replacedTn5410(Trp) or Tn5-131(Tet'), after lysogenization ofthe appropriate bacterial strain and selection of Kanrnonlysogenic segregants. Independent replacements atany given site were obtained from independently iso-lated lysogens.

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Tn5 INSERTION MUTATIONS 441

Construction of a double mutant 4insertions in kIcZ and kacP in ci '.mutant containing the insertion mutatiorTn5-131(Tetr) and lacZl24::Tn5(Kanr) in o

erated as outlined in Fig. 3. An F' la(

(nI m EKnR°S E m I

A I

R X

X R

R X

X R

FIG. 1. Map of Tn5 and its derivatitlines indicate inverted repetitions; P Mpromoter for expression ofkanamycin resstriction endonuclease cleavage sites: IBglII; III, HindIII; S, SalI; Sma, SmaI,X, XbaI. Th5-410 contains a 5,700-base-pacontaining the trp operon E and D genesthe central HindIII fragment of wild tyTn5-131 and Th5-132 contain a 2,700-basfragment from TnlO encoding tetracyclin(Tet') in opposite orientations substitutedtral BglII fragment ofwild-type Th5. In ITh5-137, the BglII Tetr fragment is inserleft component ofTn5's inverted repetitionorientations. Tn5-135 and Tn5-137 also112 deletion (represented by the openextends from the SalI site to the HpaI spairs from the end of Tn5 (Ila; R. Jorgithesis, University of Wisconsin, MadisoSchaller, personal communication).

containing 131(Tet') episome was transferred to an F- lacZ124::The double Tn5(Kanr) recipient. Lac' recombinant F' episomesas lacP204:: containing two insertion mutations in cis were ob-cis was gen- tained by selecting the Lac' Kan' Tetr recombinantscP204::Tn5- and then identifying those able to transfer the two

antibiotic resistance traits at high frequency to strainDB1512 [F- Strr A(proB-lac)].Plasmid DNA. In most cases plasmid DNAs were

Th5-WT Kan R extracted from 28 ml of cells after amplification over-Th5 40 TrpE + night with chloramphenicol, lysis in 0.3 ml of 10 mM5 Tris, 50 mM EDTA, pH 8, 50 ,ug of lysozyme per ml,

Th5-131 TetR and 0.2% sodium dodecylsulfate, precipitation of bac-terial debris and sodium dodecyl sulfate with 0.6 M

Th5- 132 TetR potassium acetate, phenol extraction, ethanol precip-itation, and resuspension of the DNA in 1 mM Tris-1mM EDTA, pH 8. In several cases 300 ml of cells were

Tn5-135 TetR extracted in the same way, and the plasmid DNA wascentrifuged to equilibrium in CsCl-ethidium bromide

Tn5-137 TeR gradienta in a Beckman 65 rotor for 48 h at 40,000rpm. The ethidium bromide was extracted with iso-

ves. Jagged propanol, and the DNA was dialyzed against 1 mMtdicates the Tris-1 mM EDTA, pH 8 (12).tistance. Re- Restriction endonuclease analyses. Plasmidr, HpaI; II, DNAs were digested with restriction endonucleasesR, EcoRI; SmaI and HinduII using conditions recommended by

sirfragment New England Biolabs. The digested DNAs were elec-s in place of trophoresed in 0.7% agarose horizontal slab gelspe-Tn5 (15). buffered with 0.16 M Tris-acetate-0.08 M sodium ace-e-pair BglII tate-0.008M EDTA, pH 8.3, at 1 to 1.5 V/cm. HindIII-e resistance digested phage A DNA from New England Biolabsfor the cen- (heated for 5 min at 70°C in 1 M ammonium acetateIh5-135 and to melt the cohesive ends) was electrophoresed in the,ted into the same gel to provide molecular-weight standards (23,in opposite 9.8, 6.6, 4.5, 2.5, 2.2, and 0.49 kilobases (18).contain the Thiogalactoside transacetylase assays. Cul-box) which tures growing exponentially in minimal 0.2% Casaminorite 186 base Acids0.2% glycerol media were harvested and assayedensen, Ph.D for thiogalactoside transacetylase as described by Ken-in, 1978; H. neil and Riezman (13), measuring the transfer of a

tritium-labeled acetyl group from labeled acetyl coen-

XCI857

I.boc Z

trp XcI857 kon1bc Z' 4 4 'Z

trploc Z' IZ

FIG. 2. Replacement of wild-type Tn5 by Tn5-410(Trp). Sites of crossing over are designated by a dashedX in the top frame and by a horizontal dashed line terminated by two vertical arrows in the second frame.The procedures used for this replacement are described in Materials and Methods.

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442 BERG, WEISS, AND CROSSLAND

T

F_ - LAC- KanR TetR--- ; l " --- PARTIAL DIPLOID

LAC4 KanR TetR

---C --- PARTIAL DIPLOIDLAC+

FIG. 3. Construction ofanF lac episome with twoTn5 elements inserted at two different sites in the lacoperon. K designates wild-type Th5. T designates theTet' derivative Tn5-131.

zyme A (0.8 Ci/mmol), which is bound by Dowexresin, to isopropyl-fl-D-thiogalactoside (IPTG), whichis not bound by Dowex resin. When needed, 10-3 MIPTG was added to induce lac operon expression atleast five generations prior to harvesting the cultures.

Special reagents. Xgal and IPTG were obtainedfrom Bachchem. 3H-acetyl coenzyme A was from NewEngland Nuclear Corp. Restriction endonucleasesSmaI and EcoRI were from New England Biolabs.

RESULTS

lac::Thn5 insertion mutants. Of the 156lac::Tn5 insertion mutants isolated as describedin Materials and Methods, 41 insertions were inlacZ, and 114 were in lacY. Since lacZ is threetimes larger than lacY (27), this distribution ofmutants indicates that Tn5 insertion is nonran-dom.

Genetic and physiological tests indicate thatone insertion, designated lacP204::Tn5, is in thelac promoter. Strains carrying this mutationmade no detectable ,-galactosidase in the ab-sence of the lac operon inducer IPTG; additionof IPTG resulted in approximately 1% of fullyinduced wild-type levels of,-galactosidase (D.E. Berg, in preparation).The effect of insertions in lacZ on lacY func-

tion was assessed by colony size on minimalmelibiose agar or by color on MacConkey meli-biose indicator agar at 410C (where lac+ coloniesare red, partially lacY are pink, and lacY arewhite) (see p. 182 in 16). Twenty-six of the 41insertions of Tn5 in lacZ were completely polaron the expression of lacY, and the other 15expressed low levels of lacY function. These twoclasses of mutants are here designated OFF andON, respectively. Independent experiments of J.Roth (personal communication) have shownthat a fraction ofTn5 insertions in the his operonof Salmonella also confer an ON phenotype.The levels of lacY expression differed among

independent ON mutants, although none ofthem fermented melibiose as well as Lac'

strains. Each lacZ::Tn5 insertion mutant gaverise to LacY+ revertants able to grow well onminimal melibiose agar. The vast majority ofthese revertants were kanamycin sensitive andthus resulted from excision of Tn5. This resultproves that the polarity determinants are withinTn5.To map the lacZ::Tn5 insertions, each mutant

was transferred by conjugation to an F- strain,which was then crossed with a series of F'lacZdeletion strains (16). The results of these crossesindicated that the OFF mutations and ON mu-tations are interspersed, although the ON mu-tations are clustered near the distal end of lacZ(Table 3).Tn5 promoter responsible for distal gene

expression. Two experiments independentlyestablished that expression of genes distal to aninserted Tn5 element is due to a Tn5 promoter,not read-through transcription from the lac op-erator region. In the first experiment, we testedthe effect of the lac operon inducer IPTG onexpression of lacA, the third gene in the lacoperon. Table 4 shows that addition of IPTG toa lac+ culture resulted in a several hundred-foldinduction of thiogalactoside transacetylase overthe noninduced level. The basal noninducedlevel represents nonspecific acetylating activitiesencoded by genes outside the lac operon, sinceit was also seen in the proB-lac deletion strainDB1163. The lacZl24::Tn5 mutant selected be-cause of its ON phenotype on melibiose agarproduced approximately 1% of the level of thio-galactoside transacetylase found in fully inducedwild type; the weaker ON mutant lacZl25::Tn5

TABLE 3. Distribution ofON and OFF Th5insertion mutations in lacZa

No. of mutants in deletion interval:Mutant

A B C D E F G H I

ON 0 0 0 0 0 0 7 9 0OFF 0 0 1 7 1 2 10 3 1

a ON mutants ferment melibiose weakly on Mac-Conkey agar and grow, albeit slowly, on minimal mel-ibiose agar, whereas OFF mutants do not fermentmelibiose and do not grow on minimal melibiose agar.The insertion mutations were mapped after transfer-ring them by conjugation from the met proC HfrCstrain DB1446 in which they were generated to F-strain DB1470 (pro' met' lac+). Pro' Met' Kan' Lac-recombinants were selected and crossed with the setof F' lac deletion strains CSH13 through CSH20 (16),and the ability to form Lac' recombinants on minimallactose plates was scored. Mutations in interval A donot recombine with CSH13 and thus would be veryclose to the promoter. The insertion in interval Irecombines with CSH20 and thus is very near thelacZ-Y boundary.

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Tn5 INSERTION MUTATIONS 443

TABLE 4. Thiogalactoside transacetylase in lacZ::Tn5 insertion mutants

Thiogalac-MelibiosePhenotypetosideStrain Metibion Phenation IPTG transacety-

utilization designation l~atvlase activ-itya

DB1446 Lac+ +++ Wild type + 100- 0.3

DB1163 Alac - Lac- + 0.2- 0.2

Z124::Tn5 ++ ON + 1.3- 1.2

Z125::Tn5 + Weak ON + 0.6- 0.5

Z109::Tn5 - OFF + 0.3- 0.3

Lac+ Kan' +++ Wild type + 100revertant of - 0.3Z124::Tn5

aThiogalactoside transacetylase activities are expressed asa percentage of fully induced wild-type levels and were deter-mined as described in Materials and Methods. Values are theaverage of results of two separate experiments.

produced approximately 0.5% of the inducedwild-type level. The thiogalactoside transacety-lase levels in these mutants are constitutive,since they were unaffected by lack of inducer.OFF mutants such as lacZlO9::Tn5 grown in thepresence or absence of IPTG contained no thio-galactoside transacetylase above the nonspecificlevel found in the uninduced wild type and inthe proB-lac deletion controls (Table 4).

Thiogalactoside transacetylase levels werealso measured in nine other ON lacZ::Tn5 mu-tants and seven other OFF lacZ::Tn5 mutants,grown in the absence of IPTG. The thiogalac-toside transacetylase levels in each of the ONstrains were 0.4 to 0.8% of induced wild type,intermediate between those of the highest ONstrain (lacZ124::LTn5) and the uninduced wild-type control. The thiogalactoside transacetylaselevels in each of the OFF mutants were less than0.4% of fully induced wild type, equivalent to thenonspecific background level. Of 23 lacY::Tn5mutants assayed, 9 had significant thiogalacto-side transacetylase activity, comparable to thatof the lacZ::Tn5 ON mutants, and 14 had onlybackground levels.

Proof that the promoter effect is due to Tn5was provided by a Lac' revertant ofON mutantlacZl24::Tn5. In the absence of IPTG, this re-vertant, like the ancestral Lac' strain, synthe-sized no detectable thiogalactoside transacety-lase (Table 3).A second experiment which indicated that the

ON phenotype results from a promoter associ-

ated with Tn5 is based on the phenotype ofstrains containing an insertion of Tn5-131(Tetr)in the lac promoter upstream from the ONinsertion mutation lacZl24::Tn5. Each of ninelacP::Tn5-131(Tetr)-lacZl24::Tn5 double mu-tant recombinants, isolated as described in Ma-terials and Methods, expressed the ON pheno-type characteristic of the lacZl24::Tn5 singlemutant. Since insertions at the lacP204 siteresulted in a nearly complete loss of lac pro-moter function (Berg, in preparation), this resultreinforces the conclusion that the ON phenotypeis due to a promoter associated with Tn5, not toread through from the lac operon promoter.

If the ON phenotype of insertion mutationssuch as lacZl24::Tn5 were due to Tn5's orienta-tion in the operon, by analogy with IS2 and TnA(10, 20, 26), then reversal of the orientation ofTn5 in the lacZl24::Tn5 mutant should changeits phenotype from ON to OFF. To reverse theorientation of Tn5 at the lacZ124 site, we re-placed the Tn5-410(Trp) element at this site instrain DB1508-124 with wild-type Tn5. This wasaccomplished by selecting for a pair of crossoversbetween the components of the inverted repeti-tions of Tn5-410 present in the lac operon andwild-type Tn5 brought into the cell by a A Tn5phage (see Materials and Methods). We as-sumed that the two components of Tn5s in-verted repetitions were homologous and thuswould permit pairing for crossing over in eitherorientation as shown in Fig. 4. Twenty-five in-dependent replacements of Tn5-410(Trp) at sitelacZ124 by wild-type Tn5 were prepared. Eachofthe isolates expressed the ON phenotype char-acteristic of the ancestral lacZl24::Tn5 straineven though in half of them the orientation ofTn5 was, as expected, reversed (see below), andthus, if analogous to IS2 and TnA, should haveconferred an OFF phenotype.

Confirmation that Tn5 was present in bothorientations was obtained by recombining thelac::Tn5 alleles into the ColEl-lac plasmidpMC4 and then digesting the purified plasmidDNAs with restriction endonucleases. The selec-tion of the pMC4 lac::Tn5 recombinants wasfacilitated by the fact that the pMC4 plasmiddoes not complement lacZ::Tn5 insertion mu-tations because pMC4 lacks the terminal 50 basepairs of lacZ (7). Consequently, as outlined inFig. 5, Lac' recombinants which remain Kanrcontain pMC4 lac::Tn5 plasmids. These plas-mids were purified by DNA extraction andtransformation of the proB-lac deletion strainDB1504 and selection for Kanr.For two plasmids containing Tn5 at identical

sites, differences in the size distribution of thetwo pMC4-Tn5 fusion fragments in the SmaIdigest reflect differences in the orientation of

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444 BERG, WEISS, AND CROSSLAND

z

xAR

N P KonR

trp

x

A

VcI8UDO d

F' acZ trp

FIG. 4. Two possible orientations ITh5 replaces Tn5-410(Trp). Proceduyplacement are described in Materials

Select Loce KonR

FIG. 5. Formation ofrecombinant jplasmids. FproB+ lac::Tn5 episomferred into strain DB1544, which carriplasmid pMC4, and Lac' Kanr Tetwere selected as described in the text.

Tn5. Figure 6A shows representapMC4 lacZl24::Tn5 and pMC4DNAs with SmaI, an enzyme whiconce and the pMC4 plasmid twicelacZl24::Tn5 insertion mutant gavtion pattern I, as did 5 of 11lacZl24::Tn5 replacements testeding 6 gave pattern II. At the lacP2replacements gave pattern I and ZII.To show that neither replaceme

bination with the pMC4 plasmidsertion sites of Tn5 elements, theTn5 plasmids were digested withcleaves Tn5 at identical sites in eaof the inverted repetition (Fig.

cleaves pMC4 once. The sizes of the HindIIIfragments of any pMC4 lac::Tn5 plasmid willdepend only on the site of Tn5 insertion, not its

ci orientation. Figure 6B confirms that neither re-placement of one Tn5 element by another nor

r recombination between the lac region on an F'episome and the pMC4 plasmid changes the Tn5insertion site.Taken together, the SmaI and the HindIII

digests prove that Tn5 has been placed in bothpossible orientations at two sites (Z124 and

NJ P204) separated by approximately 3 kilobases(see Fig. 7). The approximately equal numbersof replacements in orientations I and II indicate

y that Tn5s inverted repetitions are strongly ho-when wild-type mologous, so that the orientation of the centralres for this re- region ofTn5 is randomized by the replacementsand Methods. described.

Examination of the phenotype of strains con-taining the Tn5 substitution derivatives Tn5-

pMC4 Z_ 131(Tetr), Tn5-132(Tetr), and Tn5-410(Trp)(Fig. 1) provides independent evidence that theON phenotype is not due to a determinant in

locZ:: Tn5 Tn5's central region. In total, 15 independentreplacements of wild-type Tn5 at ON sitelacZ124 by each of these three substitution ele-ments were prepared. There was no detectablechange in the ON phenotype in any of the re-sulting 45 isolates.

Wild-type Tn5 was also replaced by Tn5-131(Tetr), Tn5-132(Tetr), and Tn5-410(Trp) in eachof three lacZ::Tn5 OFF mutants. None of these

pMC4 Z ::Tn 5 replacements led to any increase in lacY expres-sion.Localization of the promoter to within

lOC+ 186 base pairs of the end of Tn5. To furtherpMC4-lac::Tn5 map the putative Tn5 promoter responsible fores were trans- the ON phenotype, we replaced Tn5-410(Trp)ces the Tetr lacZ at site lacZ124 with Tn5-135(Tetr) and withrecombinants Tn5-137 (Tetr). These elements contain a Tetr

fragment inserted between the kanr gene and itspromoter (see Fig. 1), which results in a polar

tive digests of block on expression of kanr (a), and also a dele-lacP204::Tn5 tion which extends from the Sall site in theh cleaves Tn5 middle of Tn5 to the HpaI site 186 base pairsThe original from the end of the right component of the

{e SmaI diges- inverted repetition (see Fig. 1). Each of 50 in-independent dependent replacements of Tn5-410(Trp) at site

1. The remain- lacZ124, 27 by Tn5-135 and 23 by Tn5-137, gave!04 site, 9 of 14 strains with an ON phenotype indistinguishable5gave pattern from the parental lacZ124::Tn5-410 strain and

from the ancestral lacZl24::Tn5 wild-typent nor recom- strain. The Tn5-112 parent of Tn5-135 and Tn5-affects the in- 137 replaces Tn5-410 in both orientations at> pMC4 lacZ:: equal frequency (Berg, in preparation). Thus,YindIII, which this set of 50 strains contains Tn5-135 and Tn5-ch component 137 elements in both possible orientations, and

1) and also consequently, we conclude that the promoter

F'lac

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Tn6 INSERTION MUTATIONS

C\ I C'

<c\J\

I

I

b ,

A SmaI

FIG. 6. (A) Agarose gel electrophoresis of SmaI digests of the wild-type pMC4 plasmid (designated lac+)and representative pMC4 plasmids containing Tn5 elements at site P204 (the lac promoter) and Z124 (nearthe distal end of lacZ). The fragment sizes in kilobases (estimated using the HindIII digest ofphage A DNAas standards) are 12.0, 4.9, 5.5 and 11.2, 5.9, 5.5 from lacZl24::Tn5 orientations I and II, respectively; 8.4,8.8, 5.5 and 8.1, 9.6, 5.5 from lacP204::Tn5 orientations I and II, respectively; and 11.0 and 5.5 from thePMC4 parental plasmid. (B) Agarose gel electrophoresis of HindIII digests ofpMC4::Tn5 plasmid DNAs.Estimated fragment sizes are 10, 8.8, and 3.5 kilobases from lacZl24::Tn5, and 13.5, 6.4, and 3.5 kilobasesfrom lacP204::1n5.

4 11.2Z 124 i-

Z124 I

Z204 II

Z204 I

12

8.1 p ;

8.4

FIG. 7. Map ofSmaI sites inpMC4:Constructed using data from Fig. 6.,sites are indicated by vertical arrowsmoter is indicated by P.

responsible for the ON phenotyjlocated more than 186 base pairsof Tn5.

DISCUSSIONThe results presented here esta

5_9 44 sertions of the kanamycin-neomycin resistancetransposon Tn5 in the lac operon of E. coli

4 5 04

interrupt the expression of distal genes. In ap-d proximately two-thirds of the lac::Tn5 insertion

mutants, there is no detectable expression of96 distal genes (the OFF phenotype). In the re-

maining one-third, a promoter associated with8.8 t Tn5 results in low but variable levels of expres-

sion of distal genes (the ON phenotype).:Th5plasmids. The hypothesis that the Tn5 promoter de-SmaI cleavage tected in the ON mutants is in the central regionthe kanr pro- ofTn5 and is evident only when the Tn5 element

is in one orientation was disproved by showingthat neither (i) changing the orientation of the

pe cannot be central region of a wild-type Tn5 element norfrom the end (ii) replacing the central region of wild-type Tn5

affects the phenotypes of either the ON or theOFF insertion mutants. These experiments es-tablish that none of the sequences contained in

blish that in- the central region of Tn5 determine the ON

C;

I I

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445VOL. 142, 1980

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446 BERG, WEISS, AND CROSSLAND

phenotype and suggest that the promoter re-sponsible for the ON phenotype is within 186base pairs of the end of Tn5.Our data are compatible with each of the

following three possibilities. (i) A promoterwithin 186 base pairs of the end of one compo-nent of Tn5's inverted repetition is not presentin the other component, and hence the orienta-tion of the last 186 base pairs determineswhether the ON or OFF phenotype is expressed.(ii) The reading frame of the Tn5 insertion de-termines whether a promoter active in all Tn5insertions will be detected by its ability to stim-ulate the expression of distal genes. If so, two-thirds of the insertions would be OFF, and one-third of the insertions would be ON, just asobserved. (iii) A promoter is created at the junc-tion of Tn5 and certain bacterial sequences.Short duplications which create new sequenceshave been shown to create new promoters inphage A (17) and in IS2 (9, 23). Further experi-ments are required to distinguish between thesealternatives.

ACKNOWVLEDGMEENTWe are grateful to C. M. Berg, M. Calos, N. Franklin, D.

Kennell, R. Jorgehsen, J. Roth, and J. Shapiro for bacterialstrains and stimulating discusdons, and to M. Schmidt-Gen-genbach for expert technical assistance.

This work was supported by Public Health Service grant 5ROI AI 14267 to D. B. from the National Institute of Allergyand Infectious Diseases.

LITERATURE CITED1. Berg, D. E. 1974. Genetic evidence for two types of gene

arrangements in new Adv plasmid mutants. J. Mol. Biol.86:59-68.

2. Berg, D. E. 1977. Insertion and excision of the transpos-able kanamycin resistnce determinant Tn5, p. 205-212.In A. I. Bukhari, J. A. Shapiro and S. L. Adhya (ed.),DNA insertion elements, plasmids, and episomes. ColdSpring Harbor Pres, Cold Spring Harbor, N.Y.

3. Berg, D. E., J. Davies, B. Allet, and J. D. Rochaix.1975. Transposition of R factor genes to bacteriophageA. Proc. Natl. Acad. Sci. U.S.A. 72:3628-3632.

4. Berg, D. E., and M. Drummond. 1978. Absence ofDNAsequences homologous to transposable element Tn5(Kan) in the chromosome of Escherichia coli K-12. J.Bacteriol. 136:419-422.

5. Berg, D. E., and J. Gallant. 1971. Tests of reciprocalityin crosing over in partially diploid F' strains of Esche-richia coli. Genetics 68:457-472.

6. Berg, D. E., D. A. Jackson, and J. E. Mertz. 1974.Isolation of a A dv plasmid carrying the bacterial galoperon. J. Virol. 14:1063-1069.

7. Calos, M., L Johnsrud, and J. H. Miller. 1978. DNAsequence at the integration sites of the insertion ele-ment ISI. Cell 13:411-418.

8. Campbell, A., D. Berg, D. Botatein, E. Lederberg, R.Novick, P. Starlinger, and W. Szybalski. 1979. No-menclature of transposable elements in prokaryotes.Gene 5:197-206.

9. Ghosal, D., and H. Saedler. 1978. DNA sequence of themini-insertion IS2-6 and its relation to the sequence of

J. BACTERIOL.

IS2. Nature (London) 275:611-617.10. Heffron, F., C. Rubens, and S. Falkow. 1975. Translo-

cation of a plasmid DNA sequence which mediatesampicillin resistance: molecular nature and specificityof insertion. Proc. Natl. Acad. Sci. U.S.A. 72:3623-3627.

11. Jorgensen, R. A., D. E. Berg, B. Allet, and W. S.Reznikoff. 1979. Restriction enzyme cleavage map ofTnlO, a transposon which encodes tetracycline resis-tance. J. Bacteriol. 137:681-685.

11a.Jorgensen, R. A., S. J. Rothstein, and W. S. Rezni-koff. 1979. A restriction endonuclease cleavage map ofTn5 and location of a region encoding neomycin resis-tance. Mol. Gen. Genet. 177:65-72.

12. Kellenberger-Gujer, G., E. Boy de la Tour, and D. E.Berg. 1974. Transfer of the A dv plasmid to new bac-terial hosts. Virology 68:576-585.

13. Kennell, D., and H. Riezman. 1977. Transcription andtranslation initiation frequencies of the E. coli lac op-eron. J. Mol. Biol. 114:1-21.

14. Kleckner, N., J. Roth, and D. Botatein. 1977. Geneticengineering in vivo using translocatable drug resistanceelements: new methods in bacterial genetics. J. Mol.Biol. 116:125-159.

15. Meyer, R., G. Boch, and J. Shapiro. 1979. Transpositionof DNA inserted into deletions of the Tn5 kanamycinresistance element. Mol. Gen. Genet. 171:7-13.

16. Miller, J. H. 1972. Experiments in molecular genetics.Cold Spring Harbor Pres, Cold Spring Harbor, N.Y.

17. Mozola, M. A., D. L. Friedman, C. L Crawford, D. L.Wolff, H. Shimatake, and M. Rosenberg. 1979. Mu-tations reducing the activity of c17, a promoter of phageA formed by a tandem duplication. Proc. Natl. Acad.Sci. U.S.A. 76:1122-1125.

18. Murray, K., and N. Murray. 1975. Phage lambda recep-tor chromosomes forDNA fragments made with restric-tion endonuclease III of Haemophilus influenzae andrestriction endonuclease I of Escherichia coli. J. Mol.Biol. 98:551-564.

19. Pilacinski, W., E. Mosharaafa, R. Edmundson, J.Zissler, M. Fiandt, and W. Szybalskli. 1977. Insertionsequence IS2 associated with int-constitutive mutantsof bacteriophage lambda. Gene 2:61-74.

20. Saedler, H., H. J. Reif, and N. Davidson. 1972. IS2, agenetic element for turn-offand turn-on of gene activityin E. coli. Mol. Gen. Genet. 132:265-289.

21. Schaller, H. 1978. The intergenic region and the originsfor filamentous phage DNA replication. Cold SpringHarbor Symp. Quant. Biol. 43:401-408.

22. Shaw, K. J., and C. M. Berg. 1979. Escherichia coliauxotrophs induced by insertion of the transposableelement Tn5. Genetics 92:741-747.

23. Sommer, H., J. Cullum, and H. Saedler. 1979. IS2-43and IS2-44: new alleles of the insertion sequence IS2which have promoter activity. Mol. Gen. Genet. 175:53-56.

24. Starlinger, P., and H. Saedler. 1976. IS elements inmicroorganisms. Curr. Top. Microbiol. Immunol. 75:111-152.

25. Vogel, H. J., and D. M. Bonner. 1956. Acetylornithinaseof Escherichia coli: partial purification and some prop-erties. J. Biol. Chem. 218:97-106.

26. Weinstock, G. M., M. M. Susskind, and D. Botatein.1979. Regional specificity of illegitimate recombinationby the translocatable ampicillin resistance element Tnlin the genome of phage P22. Genetics 92:685-710.

27. Zabin, I., and A. Fowler. 1978. f8-Galactosidase, thelactose permease protein, and thiogalactoside trans-acetylase, p. 89-121. In J. Miller and W. S. Reznikoff(ed.), The operon. Cold Spring Harbor Press, ColdSpring Harbor, N.Y.

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.org/D

ownloaded from