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Chapter 29 : Transposon Mutagenesis

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Abstract:

Transposon mutagenesis has been used to clone genes, to construct reporter gene fusions, to construct correlated physical and genetic maps of cloned DNA segments, to map entire bacterial genomes by pulsed-field gel electrophoresis, to construct conditional mutations with portable promoters, to introduce desired origins of conjugal transfer or replication into chromosomes and plasmids of interest, and to determine the sequence of large DNA regions without the need of sub-or deletion cloning. In this chapter, a subset of these applications and the corresponding experimental protocols are described. Region-directed mutagenesis usually involves the isolation of Tn5 insertion mutations in genes cloned into plasmids in , followed by the characterization of the mutant phenotype of this (heterologous) host or the organism of origin of the cloned genes after the reintroduction of the Tn5-mutated loci by gene replacement. The chapter focuses on the use of bacteriophage λ and conjugable narrow-host-range or conditionally replicating plasmid vectors to deliver Tn5 and its derivatives and describes experimental protocols that are commonly used to carry out the random mutagenesis of (predominantly gram-negative) organisms.

Citation: Rossbach S, De Bruijn F. 2007. Transposon Mutagenesis, p 684-708. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch29

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FIGURE 1

Structure of the transposable element Tn. Stippled bars represent the insertion sequences ISL (left) and ISR (right), present as terminal inverted repeats in Tn. Gene symbols: , coding region for the transposase; , coding region for the inhibitor; UAA, stop codon responsible for the truncated versions of the transposase and the inhibitor in ISL; p, promoter for the transcript of the operon coding for resistance to three antibiotics (, neomycin or kanamycin resistance; , bleomycin resistance; , streptomycin resistance). Abbreviations for restriction enzymes: Ba, BamHI; Bg, BglII; H3, HindIII; Hp, HpaI; No, NotI; Ps, PstI; Pv, PvuII; Sa, SalI; Sm, SmaI; Xh, XhoI.

Citation: Rossbach S, De Bruijn F. 2007. Transposon Mutagenesis, p 684-708. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch29
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Citation: Rossbach S, De Bruijn F. 2007. Transposon Mutagenesis, p 684-708. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch29
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Tables

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TABLE 1

Common bacterial transposable elements and their characteristics

Abbreviations used for resistance phenotypes are as follows: Ap, ampicillin; Km, kanamycin; Nm, neomycin; Bm, bleomycin; Sm, streptomycin; Tp, trimethoprim; Sp, spectinomycin; Cm, chloramphenicol; Tc, tetracycline. Bm and Sm phenotypes associated with Tn are expressed only in certain nonenteric bacteria.

Citation: Rossbach S, De Bruijn F. 2007. Transposon Mutagenesis, p 684-708. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch29
Generic image for table
TABLE 2

Tn derivatives used for the creation of gene fusions

Citation: Rossbach S, De Bruijn F. 2007. Transposon Mutagenesis, p 684-708. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch29
Generic image for table
TABLE 3

Tn derivatives carrying portable promoters, alternative marker genes, and useful sites

Amy, ability to degrade starch.

Bgl, ability to ferment cellobiose.

, delta endotoxin gene from .

, gene for resistance to arsenite; vector designated pUT/Ars by Herrero et al. ( ).

, gene for resistance to the herbicide bialaphos; vector designated pUT/PTT by Herrero et al. ( ).

, gene for resistance to mercuric salts and organomercurial compounds; vector designated pUT/Hg by Herrero et al. ( ).

Citation: Rossbach S, De Bruijn F. 2007. Transposon Mutagenesis, p 684-708. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch29
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Citation: Rossbach S, De Bruijn F. 2007. Transposon Mutagenesis, p 684-708. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch29
Generic image for table
Untitled

Citation: Rossbach S, De Bruijn F. 2007. Transposon Mutagenesis, p 684-708. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch29
Generic image for table
Untitled

Citation: Rossbach S, De Bruijn F. 2007. Transposon Mutagenesis, p 684-708. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch29

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