tn5 induced auxotrophy

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ESCHERZCHZA COLI K-12 AUXOTROPHS INDUCED BYINSERTION OF THE TRANSPOSABLE ELEMENT Tn5

KAREN J. SHAW AND CLAIRE M. BERG1Biological Sciences Group (BoxU-l31), The Univers it y of Connecticut,Storrs, Connecticut 06268

Manuscript received October 5, 1978Revised copy received March 7, 1979

ABSTRACTThe siies of insertion of the transposable kanamycin-neomycin resistance-

determining element, Tn5, in the E . coli K-I2 chromosome were assessed ina collection of over 300 auxotrophs. Although mutations in at least 45 dif-ferent cistrons were obtained, the distribution of insertion sites was not com-pletely random: proA o r proB; cysG; and c y sH , cysD or cysC mutants werefound in excess.

TRONGLY polar mutations are often due to insertion of a segment of DNAinto a gene, interrupting its continuity and disrupting transcription of distal

genes in the same operon (STARLINGERnd SAEDLER976). DNA insertion ele-ments range from small (800 to 1400 base pairs) IS elements carrying no knowngenes to complex phages such as Mu and A. lntermediate in size and complexityare the transposable (Tn) elements, which carry antibiotic resistance determi-nants and possibly additional genes unrelated to inser tion function. Mutationsdue to the insertion of each class of element have been obtained in Escherichiacoli K-12 (TAYLOR963; JORDAN,AEDLERnd STARLINGER968; SHAPIRO969;SHIMADA,WEISBERGnd GOTTESMAN972; KLECKNERt al. 1975; BERG1977).While there is no straightforward way to select fo r IS element-induced insertionmutants, Tn element-induced mutants may be readily selected because of theantibiotic resistance determinants that they carry.

We have isolated over 300 E. coli mutants induced by the kanamycin-neomycin resistance-determining element, Tn5, to assess whether the elementinserts preferentially at certain sites o r randomly throughout the chromosome,and to isolate mutants f o r use in further studies. These insertion mutants areuseful f o r studies of operon polarity, internal promoters. indirect suppression andnull activity mutants.

MATERIALS AND METHO DSStrains: Insertion mutants were induced in Escherichia coli K-12 strain W3110(thy) using

phage cl857 b221 (deleted for 1 at t ) carrying the transposable element, Tn5 (X::Tn5) (D.BERQ t al . 1975). The F strains were obtained from the E . coli Genetic Stock Center, YaleUniversity, New Haven, Connecticut.' o whom reprint requests should be sent.

Genetics 9 2 741-747 July, 1979


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742 K . J.SHAW A N D C. M. BERGMedia: Tryptone broth containing 8 g/l tryptone, 5 g/l NaCI, 2 g/l maltose, and 10 mg/l

thymine was used for h::Tn5 infection of E . coli . The complex medium contained 5 g/l sodiumcitrate, 1 g/l glucose, 10 g/l tryptone, 5 g/1 yeast extract, 10 mg/l thymine and 15 g/l agar.In some experiments, the complex medium was supplemented with 40 mg/l DL-glutamate, 50mg/l DL-diaminopimelic acid, and 40 mg/l DL-alanine in order to detect cell-wall mutants.Medium E (VOGELnd BONNER 956) plus 5 g/l glucose, 10 mg/l thymine and 2 mg/ l thiaminewas employed as the minimal medlum. In some experiments 15 g/1 sodium succinate was sub-stituted for glucose in the minimal medium.Mutagenesis: Cells grown overnight a t 37" in tryptone broth plus 0.2% maltose were centri-fuged and resuspended at about 5 x 108/ml in 0.01 M MgSO,. Phage were added at a finalmultipl icity of infection of five to ten, and the mixture was incubated f o r 15 min at 30". Themixture was then diluted ten-fold with tryptone broth lacking maltose and aerated at 30" for30 min to allow for phenotypic expression of antibiotic resistance. The cells were spread on freshplates containing complex agar plus 10 pg/ml neomycin sulfate (Sigma), and incubated for48 hours at 30". The colonies were replica plated to minimal medium, and the replicas wereallowed to grow for 24 hours at 30". Auxotrophs were purified and tested for growth require-ments at 3 7 " .Mutant characterization: About l o 7 cells of each mutant were spread on a glucose minimalplate and tested by spotting drops of amino acid, vitamin and purine plus pyrimidine pools(individual supplements were obtained from Sigma), and also casamino acids (Difco) on eachplate. After 24 to 48 hr incubation at 37", the plates were scored for growth around or betweenthe pools. Mutants were retested fo r growth on plates on which the appropriate individual com-ponents had been spotted.

After the mutants had been classified by growth requirement, they were further charac-terized by their abili ty to crossfeed or be crossfed by other members of the same phenotypicclass, their ability to grow on biosynthetic intermediates and, in some cases, by F' complementa-tion mapping or enzyme assays.

RESULTS AN D DISCUSSIONTh e kanamycin-neomycin resistance-determining transposable (Tn) element,

Tn5, transposes from lambda to the Escherichia coli chromosome at a frequencyof about About 1% of these antibiotic-resistant (Neo') isolates are auxo-trophic (D. BERG1977; this work). Since there is no evidence for multiple inser-tion of Tn5 during transposition (D. BERG, ersonal communication), the proba-bility of obtaining a strain with two Tn elements inserted is negligible ( c a . lo-.).Therefore Tn5 is an effective single-site mutagen. Tn5 inserts into the chromo-some with a higher frequency than other Tn elements (KLECKNER, OTHandROTSTEIN 1977), and most insertion mutants are stable, with a true revertantfrequency of i o-. (D. BERG1977).

In this study, 305 Tn5-induced auxotrophs were isolated after infection of E .coli with A: :Tn5 and selected on complex medium containing neomycin. Theywere identified by their inability to grow when replica plated onto glucose mini-mal medium. Since the auxotrophs were isolated without enrichment, they reflectthe natural distribution of insertion sites detectable under the conditionsemployed.

An accurate estimate of the reversion frequency could not be obtained formany of the auxotrophs. In some cases, the number of revertants was not propor-tional to the number of cells plated (competitive suppression: RYAN 1953) andin other cases indirectly suppressed revertants outnumbered true revertants.


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Tn5-INDUCED AUXOTROPHS 743The most conspicuous mutants of the latter type were the proAB::Tn5 mutants,where about 80% of the revertants were still proAB::Tn5. Since spontaneousproAB mutants are known to revert preferentially via forward mutation at argD(C. BERG nd ROSSI1974), this observation was not surprising.Nutritional requirements of 274 of the 305 auxotrophs were determined on thebasis of growth response to various metabolites. Mutants with a common require-ment could be further subdivided using a combination of crossfeeding behavior,growth response on biosynthetic intermediates, F complementation testing and,in some cases, enzyme assays (Table 1, Figure 1). One mutant, which did notrespond to any supplement o n glucose minimal medium, was able to grownormally on succinate minimal medium, but was inhibited if glucose was addedto the plate. Thirty mutants could not be characterized with the tests employed.

TABLE 1Tn5-induced auxotrophs in E. coli K - I 2 W3110 (thy) *

Me,:hod,of Site ofSymbol identification+ insertlon Number T o t a l Percent

Dap

GlYGuaHis

I l u

Leu

BCEGHI

unidentifiedB

A or Baunidentified

I or JH , D or C

EG

A o r Eunidentified

AA or B

DEG

unidentifiedACDE

unidentified

34541223318534

22112143144972

16

213

4

87

12

2 216

6.91 o

1.3

28.5

0.70.70.3

3.9

7.25.2


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7444 K. J . SHAW AND C. M. B E R GTABLE 1-Continued

Method,of Site ofSymbol identificauont insertion Number Total Percent

LYSM e t

PheProPur$

PYr

Ser

T h rTrP

UnclassifiedGlucose sensllTotal

AEJ, B, F, L or M

unidentifiedA

A o r BCAc,G, 1EF

J, D, HASB

C o r DF

unidentifiedB

unidentifiedAB or CABD

unidentified

20 6.6

2 2 0.7437 14 4.64 4 1.3

192 21 6.91921762911 19 6.226 8 2.634 7 2.32223 9 3.0

30 30 9.81 1 0.3

305 99.8* Gene designations as in BACHMANN, ow and TAYLOR1976), except for the C y n mutantswhich are as in QURESHI, MIT H nd KINGSMAN1975).j-Cistron assignments were based upon: (a) growth on intermediates; (b) crossfeeding; (c) Fcomplementation; (d) enzyme assays; (e) cotransduction (H . LIEBKE, ersonal communica-tion);and ( f ) intermediate accumulation (D. BERG, ersonal communication).$ C a r A or B.Revertants accumulate in most of these strains i f they are stored in broth.This mutant will grow on succinate as a carbon source, but it i s inhibited by glucose.The argZ mutants were unexpected because wild-type E . coli has duplicate

genes coding for isozymes of ornithine carbamoyltransferase (BACHMANN, owand TAYLOR976) . Since single-step mutants defective in this enzymatic activityare not generally obtained, it appeared probable that W 3 1 1 0 (thy) was eitherargF or argl . F' strains carrying either argF+ o r argl+ yielded Arg+ recombi-


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Tn5-INDUCED A U X O T R O P H S 745U

f m

'2.F

FIGURE.-Map locations of the mutations identified in Table 1. The map positions areaccording to BACHMANN, ow and TAYLOR1976).nants with both Tn5-induced mutants. Since our strain of W 3 l l O ( t h y ) is alsoZacA (D. BERGpersonal communication), we assume that this strain carries anargF-lacAdeletion and that Tn5 is inserted into argl.

It is clear that Tn5 inserts at many sites in the E . coli chromosome (Table 1,Figure 1 ) . However, insertion is not completely random because several classesof auxotrophs were obtained in excess. Cysteine-requiring mutants, fo r example,comprised 2 9 % of the total, with two genes or gene clusters accounting for theexcess: 7% of all the auxotrophs isolated were cysG and 17% were cysH, cysDor cysC. Insertion into other cys sites was obtained at low frequencies. proA orproB mutants were also obtained with above average frequencies (6%). A non-random distribution of Tn5 insertion sites in lac2 us. lacy has been foundpreviously (D. BERG,personal communication).No DNA insertion element has been found to insert into the bacterial chromo-some randomly. A (deleted fo r att) inserts preferentially into pro in E . coli, butalso inserts into other genes at < 3% of the pro frequency (SHIMADA, EISBERGand GOTTESMAN9 7 3 ) . Mu induces an excess of pur, pro and met mutants inE . coli (TAYLOR9 6 3 ) . The relative frequencies of TnZO-induced auxotrophs inS . typhimurium differ from one report to another (KLECKNERt al. 1975; KLECK-NER, ROTH nd BOTSTEIN 9 7 7 ) . The distribution of phenotypic classes in a smallsample of Tn5-induced auxotrophs obtained in a previous study with E . coli(KLECKNER,OTHand BOTSTEIN 977) differs from our findings (Table 1 ) , but


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746 K. J. SHAW AND C. M. BERGthe major discrepancy between the two sets of data (the absence of pro mutantsin that study) is due to the fact tha t a pro auxotroph had been used as the recip-ient for Tn5 transposition (H. LIEBKE, ersonal communication).

Th e patterns of insertion of Tn5, and TnlO are different, but comparable, ifmutants are classified only with respect to required metabolite. However, whenmutants within a gene are mapped, the Tn5 and TdO patterns are dissimilar.For example, in the lac2 gene of E. coli, only 5% of the Tn5 insertions are notseparable by recombination (D. BERG1977), while 86% of the TnlO insertionsfall in one small region (FOSTER 977). Insertion of Tn5 seems, therefore, morerandom than insertion of T d O .

Transposable elements carrying drug-resistance determinants provide a simpledirect selection procedure for the isolation of viable insertion mutants withoutrecourse to indirect selection procedures or concern about the isolation of multiplemutations. In addition, since the transposition of a Tn element into a geneeliminates all detectable gene function and has a polar effect upon the expres-sion of operator-distal genes (D. BERG 977; KLECKNERt al. 1975; HEFFRON,RUBENS nd FALKOW977), insertion mutants can be used in studies in whichthe absence of a gene product is essential and in the detection of internal promo-ters. Using crossfeeding, we have been able to detect the internal promoterspreviously described in the argCBH (ELSEVIERSt al. 1972; JACOBY972), andhis (ATKINS nd LOPER 970) operons, as well as a previously overlooked inter-nal promoter in the iZuGEDA operon (C . BERG et al., 1979). Additionally.the mutants isolated will be invaluable in the study of indirect suppression be-cause true revertants, which lose Tn5 (D. BERG19771, may be readily distin-guished from indirectly suppressed revatants, which retain Tn5 (unpublished),on the basis of antibiotic resistance.

Since transposable elements insert into a large number of different sites, manymutations which were previously difficult to select now may be manipulatedeasily by transduction or conjugation because of linkage of the mutation to theantibiotic-resistance phenotype. In addition, since the possible sites of insertiondiffer from element to element, it is useful t o have several well-characterizedcollections of mutants induced by the insertion of different transposable elements.

We are indebted to D. BERG nd H. LIEBKE or many stimulating discussions, critical readingof the manuscript and for characterizing a few of the mutants. B. BACHMANNindly providedthe F strains used. We are also grateful to B. BACHMANNnd K. B. Low f o r conversationsregarding a r g l and argF .

This work was supported by Public Health Service grant AI-14276 from the National Insti-tute of Allergy and Infectious Diseases and by National Science Foundation grant PCM76-81615.K. J. SHAWwas supported by Genetics Training grant lT32GM07584 from the National Instituteof General Medical Sciences.

LITERATURE CITEDATKINS,J. F. and J. C. LOPER,1970BACHMANN,. J., K. B. Low and A. L. TAYLOR,976

Transcription initiation in the histidine operon of Sal-Recalibrated linkage map of Escherichia

monella typhimurium. Proc. Natl. Acad. Sci. U.S. 65: 925-932.coli K-12. Bacteriol. Rev.4.0: 16-167.


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Tn5-1NDuCED AUXOTROPHS 747BERG,C. M. and J. J. ROSSI,1974BERG,C. M., K. J. SHAW, . VEN DER nd M . BORUCKA-MANKIEWICZ, 1979

Proline excretion and indirect suppression in Escherichiacoli and Salmonella typhimurium. J. Bacteriol. 118: 928-939.

Physiological char-acterization of polar Tn5-induced isoleucine-valine auxotrophs in Escherichia coli K-12:Evidence for an internal promoter in the ilvGEDA operon. Genetics 93 (In Press).

Insertion and excision of the transposable kanamycin resistance determinantTn5. pp. 205-212. In: DNA Insertion Elements, Plasmids and Episomes. Edited by A. I.BUKHARI, J. A. SHA PIRO nd S . L. ADHYA. old Spring Harbor Laboratory, Cold SpringHarbor, New York.

Transposition of R factor genes tobacteriophage A. Proc. Natl. A cad. Sci. U.S. 72: 3628-3632.

Control regionswithin the argECBH gene cluster of Escherichia coli K12. Mol. Gen. Genet. 117: 349-366.

Insertion of the tetracycline resistance translocation unit TnlO in the lacoperon of Escherichia coli K12. Mol. Gen. Genet. 154: 05-309.Transposition of a plasmid DNA sequencewhich mediates ampicillin resistance: general description and epidemiologic considerations.pp. 151-160. In: DNA Insertion Elements, Plasmids, and Episomes. Edited by A . I. BURHARI,J. A. SHAPIROnd S. L. ADHYA . old Spring Har bor Laboratory, Cold Spring Harbor, NewYork.

JACOB Y, . A., 1972 Control of the argECBH cluster in Escherichia coli. Mol. Gen. Genet. 117:337-348.JORDAN,E., H. SAEDLERnd P. STARLINGER,96 8 00 nd strong-polar mutations in the galoperon are insertions. Molec. Gen. Genet. 102: 353-363.KLECKNER, ., R. K. CHAN,B. K. TYE nd D. BOTSTEIN,975 Mutagenesis by insertion of a

drug-resistant elem ent carrying an inverted repetition. J. Mol. Biol. 97: 561-575.KLECKNER,., J. ROTH nd D. BOTSTEIN,977 Genetic eng inee ring in vivo usin g translocatabledrug-resistance elements: new methods in bacterial genetics. J. Mol. Biol. 114: 125-159.QURESHI,M. A., D. A. SMITHand A. J. KINGSMAN,97 5 M utants of Salmonella typhimuriumresponding to cysteine or methionine: their nature and possible role in the regulation ofcysteine biosynthesis. J. Gen. Microbiol. 89: 353-370.

BERG,D. E., 1977

BERG,D. E., J. DAVIES, . ALLETand J-D. ROCHAIX,97 5ELSEVIERS,., R. CUNIN,N. GLANSDORFF,. BAUMBERGnd E. ASHCROFT,97 2FOSTER,. J., 1977HEFFRON,., C. RU BEN S nd S. FALKOW,97 7

RYAN,F. D., 1953SHAPIRO,. A., 1969 Mutations caused by the insertion of genetic ma terial int o the galactoseoperon of Escherichia coli. J. Mol. Biol. 40: 3-105.SHIMA DA, ., R. A. WEISBERGnd M. E. G O T T E S M ~ N ,97 2 Prophage lambda at unusual

chromosome locations I. Location of the secondary attachment sites and the properties ofthe lysogens. J. Mol. Biol. 63: 483-503. -, 1973 Prophage lambda at unusualchromosome locations 11. Mutations induced by bacteriophage lambda in Escherichia coliK-12. J. Mol. Bi d. 80: 297-314.STARLINGER,. and H. SAEDLER, 976 IS-elements in microorganisms. Current Topics inMicrobiology and Imm unology 75: 111-152.TAY LOR , . L., 1963 Bacteriophage-induced mutation in Escherichia coli. Proc. Natl. Acad.Sci. U.S. 50: 1043-1051.VOGEL,H. J. and D. M . BONNER , 956 Acetylornithinase of Escherichia coli: partial purifica-tion a nd some properties. J. Biol. Chem. 218: 97-106.

Corresponding editor: G. MOSIG

Competitive suppression of prototrophs. Nature 171: 400-401.

tn5 induced auxotrophy

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