[2] use of antibiotic‐resistant transposons for mutagenesis 11

[2] Use of Antibiotic‐Resistant Transposonsfor Mutagenesis

By STANLEY R. MALOY

Abstract

One of the greatest advances in molecular genetics has been the appli-cation of selectable transposons in molecular biology. After 30 years ofuse in microbial genetics studies, transposons remain indispensable toolsfor the generation of null alleles tagged with selectable markers, geneticmapping, manipulation of chromosomes, and generation of various fusionderivatives. The number and uses of transposons as molecular tools con-tinues to expand into new fields such as genome sciences and molecularpathogenesis. This chapter outlines some of the many uses of transposonsfor molecular genetic analysis and strategies for their use.

Introduction

Antibiotic‐resistant transposons have revolutionized bacterial genetics.Transposons provide efficient methods of constructing mutations andselectable linked genetic markers, moving mutations into new strains, facil-itating localized mutagen esis of de fined regi ons of the genome (see Chapter6), an d constr ucting fusio ns (see Chapt er 13 ). In addit ion, because multip lecopies of a transposon in a cell provide regions of genetic homology that canbe placed at any desired position, transposons can be used to select forrecombination events that yield defined deletions and duplications (seeChapter 7), insertio ns, or inver sions (Kl eckner et al., 1977 ). A brief list ofsome of the most common uses of transposons follows (modified fromKleckner et al., 1991).

1. Transposons can be inserted at a large number of sites on thebacterial chromosome. It is possible to find transposon insertions inor near any gene of interest.

2. With very rare exceptions, transposon insertions result in completeloss of function.

3. The insertion mutation is completely linked to the phenotype ofthe transposon in genetic crosses. This makes it easy to transfermutations into new strain backgrounds simply by selecting fortransposon‐associated antibiotic resistance.

METHODS IN ENZYMOLOGY, VOL. 421 0076-6879/07 $35.00Copyright 2007, Elsevier Inc. All rights reserved. DOI: 10.1016/S0076-6879(06)21002-4

1

12 transposons [2]

4. It is feasible to screen for transposon insertion mutants after low‐level mutagenesis because each mutant that inherits the transposon‐associated antibiotic resistance phenotype is likely to have onlyone insertion. The antibiotic resistance provides a selectable markerfor backcrosses to establish that the mutant phenotype is dueexclusively to one particular transposon insertion.

5. Transposon insertions in operons are nearly always strongly polaron expression of downstream genes. Thus, transposon insertionscan be used to determine whether genes are in an operon.

6. Transposons can generate deletions nearby. This provides aconvenient method for isolating adjacent deletion mutations.

7. Transposons can provide a portable region of homology for geneticrecombination. Transposon insertions can be used to constructdeletions or duplications with defined endpoints, or can serve assites of integration of other genetic elements.

8. When used as a recipient in a genetic cross, transposon insertionsbehave as point mutations in fine‐structure genetic mapping.

9. Transposon insertions can be obtained that are near but not withina gene of interest. Such insertions are useful for constructingdefined deletions and duplications, as well as for genetic mapping.

0. Special transposons can be used to construct operon or genefusions.

Transposase

The enzymes that catalyze transposition differ with respect to activityand site selectivity (Craig et al., 2002). Transposase from the commonlyused transposons Tn5 and Tn10 show clear DNA sequence specificity forthe insertion site, but the site recognized is sufficiently permissive to allowthe isolation of insertions in essentially any gene. The transposase from Mualso demonstrates less DNA sequence specificity for the insertion site.Transposase mutants can reduce the sequence specificity of insertion. Forexample, mutations in the Tn10 transposase gene tpnA have been isolatedwith relaxed site speci ficity (Ben der a nd Kleckner , 1992). Use of trans po-sase derivatives with altered site specificity can increase the site saturationof transposon insertions, and are particularly useful for isolation of raremutants.

In addition, a variety of transposons have been engineered to removethe transposase from the transposon, demanding that the transposase isprovided from a location adjacent to the transposon or in trans fromanother transposon or plasmid. Such defective mini‐transposons are veryuseful because once the insertion has been separated from the transposase,

[2] use of antibiotic‐resistant transposons for mutagenesis 13

the insertions are stable and secondary transposon events are eliminated.These mini‐transposons must contain the ends of the transposon requiredfor transposition, typically flanking a useful antibiotic‐resistance gene.

Delivery of Transposons

To be used as effective genetic tools, transposons have to be efficientlydelivered to the recipient cells. Transposition typically occurs at a lowfrequency in vivo. Therefore, it is essential to have an efficient geneticselection to isolate a collection of transposon insertions in a host. A gooddelivery system provides a selection for transposition. Various approachesare commonly used for the in vivo delivery of transposons.

Phage Delivery Systems

Phage delivery systems take advantage of a transposon insertion on aphage that is unable to lyse or lysogenize the recipient cell. For example,lambda cI::Tn10 P(Am) cannot form lysogens because the Tn10 insertiondisrupts the cI gene, and cannot grow lytically in a supo recipient becausethe P gene product is required for phage replication. A lysate of this phageis prepared on an Escherichia coli amber suppressor mutant, and then anE. coli supo recipient is infected with the phage, selecting for tetracyclineresistance (TetR) encoded by the Tn10. The resulting TetR colonies resultfrom transposition of Tn10 from the disabled phage onto the chromosome.

An analogous approach relies on the transfer of a phage carrying atransposon from one bacterial species where the phage can reproduce to aspecies where the phage can infect but cannot replicate. For example,phage P1 can efficiently infect and replicate in E. coli, and can infectMyxococcus xanthus but cannot replicate in M. xanthus. A lysate of P1carrying a transposon is grown in E. coli, and then this lysate is used toinfect M. xanthus. When M. xanthus is infected with a lysate of P1::Tn5with selection for kanamycin resistance (KanR) encoded by the Tn5, theresulting KanR colonies are results of the transposition of Tn5 from thedisabled phage onto the chromosome.

Plasmid Delivery Systems

Plasmid delivery systems take advantage of a transposon insertion on aplasmid that is unable to replicate in the recipient cell (i.e., the transposonis carried on a suicide plasmid). The plasmid can be transferred from thepermissive host to the nonpermissive host by conjugation, transformation,or electroporation, with selection for an antibiotic resistance encoded bythe transposon.

14 transposons [2]

Overexpression of Transposase in trans

The frequency of transposition can often be improved by increasingthe concentration of transposase in the cell. For example, the transposasegene(s) can be cloned from a transposon into a vector that places theirexpression under the control of an easily regulated promoter, such as thetac promoter or the ara promoter. When the transposon is introducedinto a cell producing high levels of transposase, the frequency of transposi-tion is sufficiently high that it can often be detected without a directselection.

Transposon Pools

It is often useful to isolate a collection of transposon insertions at manydifferent sites in the genome of a bacterium. A population of cells that eachcontains transposon insertions at random positions in the genome (a trans-poson ‘‘pool’’) can be used to select or screen for those cells with insertionsin or near a particular gene. For a genome the size of E. coli or Salmonellawith about 3000 nonessential genes, a transposon pool made up of approxi-mately 15,000 random insertions will represent an insertion in most non-essential genes. About 1 in 3000 of these cells will contain a transposoninsertion in any particular nonessential gene, and about 1 in 100 of thesecells will contain a transposon insertion within 1 centisome (i.e., 1 min or1% of the chromosome length) of any particular gene.

A transposon pool can be made by any transposon delivery system.Figure 1 shows how a Tn10 pool could be constructed in Salmonella.

To isolate insertions in or near a gene, cells with transposon insertionsare first selected by plating onto a rich medium containing an antibioticresistance expressed by the transposon. The resulting colonies can then bescreened for insertion mutations in a gene or insertions near a gene. Anexample of how to isolate insertions near a gene is shown in Fig. 2.

cis Complementation

Many transposases act preferentially on the DNA from which they areexpressed. Often they must be expressed at high levels to work effectivelyin transposition. Transitory cis complementation is a mechanism thatallows cis complementation for transposition of transposons deleted fortheir cognate transposase, but after transposition, the DNA encodingtransposase is degraded leaving insertions that are not capable of fur-ther transposition. This is accomplished by placing the transposase gene

Defective for lytic growthDefective for lysogenic growthP22

Tn10

Infect population of 109 cells

Select TetRTransposition results in a randomcollection of Tn10 insertions at differentsites on the chromosome of each cell

Grow P22 HT transducingphage on these cells

Random chromosomal DNA fragmentsare packaged into transducing particles withabout 1 min of DNA in each phage head

xyz abcabc def

rst uvw

hij klmqrs tuv

fgh ijk

abc def

efg hij

lmn opq

lmn opq

pqr stu

xyz abc

Transduce TetS recipientselecting TetR

Cells that acquire a Tn10 insertion byrecombination with a transducingparticle will form colonies

Screen resulting colonies for Tn10 insertions in or near any gene of interest

FIG. 1. Isolation of a random pool of Tn10 insertion mutants.

[2] use of antibiotic‐resistant transposons for mutagenesis 15

a+b+c+

a+b+c+

a+b+c+

Tn10

Tn10

aux+e+f+

aux+e+f+

aux−e+f+

( )

Select TetR

Screen for d+

Example screening for a linked Tn10 insertion linked to aux+ by replica plating

Donor = P22 grown on random pool of Tn10 insertions in aux+ strainRecipient = aux− Tets

Transduce recipientselecting TetR

(1)

(2)

Rich medium with tetracycline

Replica plate to screenfor prototrophs

Minimal medium without supplement Minimal medium with supplement

(3) Backcross Tn10 insertion to confirm link to aux+

FIG. 2. Screen for Tn10 insertions near a biosynthetic gene using a generalized transducing

phage grown on a random pool of transposon insertions. In the example, auxþ is protrotrophic

and can thus grow on minimal medium, while aux� is auxotrophic and cannot grow on

minimal medium both auxþ and aux� can grow on rich medium.

16 transposons [2]

[3] IN VIVO mutagenesis using EZ‐Tn5tm 17

adjacent to its transposon target under conditions where the transposase isrepressed. Introduction of the DNA by transduction, transformation, orelectroporation into recipient cells allows for the induction of transposasethat can then act on the adjacent transposon for transposition in the recipi-ent cell. At some point, the remaining DNA including the transposase geneis degraded by nucleases resulting in a transposon insertion that is incapableof further transposition.

References

Bender, J., and Kleckner, N. (1992). Is10 transposase mutants that specifically alter target site

specificity. EMBO J. 11, 741–750.

Craig, N., Craigie, R., Gellert, M., and Lambowitz, A. (2002). ‘‘Mobile DNA II.’’ ASM Press,

Washington, DC.

Kleckner, N., Roth, J., and Botstein, D (1977). Genetic engineering in vivo using

translocatable drug‐resistance elements. New methods in bacterial genetics. J. Mol. Biol.

116, 125–159.

Kleckner, N., Bender, J., and Gottesman, S. (1991). Uses of transposons with emphasis on

Tn10. Methods Enzymol. 204, 139–180.

[3] In Vivo Mutagenesis Using EZ‐Tn5TM

By JOHN R. KIRBY

Abstract

Epicentre Biotechnologies has developed a suite of transposon‐basedtools for use in modern bacterial genetics. This chapter highlights the EZ‐Tn5TM TransposomeTM system and focuses on in vivo mutagenesis andsubsequent rescue cloning. Many other applications and variations havebeen describ ed and are availabl e through Epi centre’s websit e at http:/ /www.epibi o.com/ .

Introduction

The EZ‐Tn5TM TransposomeTM system from Epicentre provides arapid and straightforward method for in vivo mutagenesis and target iden-tification following rescue cloning from the desired mutant. The EZ‐Tn5TM

system is based on the hyperactive Tn5 system previously described byGoryshin and Rezn ikoff (1998) .

METHODS IN ENZYMOLOGY, VOL. 421 0076-6879/07 $35.00Copyright 2007, Elsevier Inc. All rights reserved. DOI: 10.1016/S0076-6879(06)21003-6