Members of the mariner family of transposable elements are widespread in nature, and are found in eukaryotes ranging from insects to humans (Plasterk et al. 1999). Recently, naturally occurring active elements from this family have also been found in bacteria (Cassier-Chauvat et al. 1997; Larsson et al. 2005; Feng and Colloms 2007). These elements, which are members of the IS630 family of Insertion Sequences, are flanked by TA target duplications and carry single transposase genes, the products of which share sequence similarity with mariner transposases of eukaryote origin.

At present, more than 800 eubacterial genomes have been sequenced, and many more are due to be completed. The new wealth of data generated by genome projects requires deciphering. Transposons have been widely used as genetic tools that can insert randomly into microbial genomes. This system is particularly useful for the identification of new genes of unknown function. Although many bacterial transposons have been engineered to construct insertional mutagenesis systems for several bacterial species by using both in vivo and in vitro delivery methods (Polard and Chandler 1995), mariner elements offer several advantages. First of all, they do not require species-specific host factors for efficient transposition. By contrast, bacterial transposable elements require host factors to increase the efficiency of the transposition reaction. As all of the major steps of transposition are catalyzed by the mariner transposase, mariner elements do not present host range restrictions and they have been demonstrated to transpose in both eukaryotes and prokaryotes (Plasterk et al. 1999). Second, apart from the dinucleotide TA, mariner elements have no specific sequence requirements for their insertions (Lampe et al. 1996). By contrast, for example, mutagenesis with the Streptococcus faecalis transposon Tn917 is not completely random and Tn917 insertions occur at several hot spot regions of the chromosomes of Bacillus subtilis (Youngman et al. 1985), Enterococcus faecalis (Garsin et al. 2004), and Streptococcus equi (Slater et al. 2003). In addition, transformation with mariner elements usually leads to 10-fold-more mutants than transformation with the Tn917-based vector in Listeria monocytogenes (Cao et al. 2007). There has therefore been a great deal of interest in developing transposons from the mariner family for applications in bacterial genetics.

Random transposon mutagenesis

Transposon-based mutagenesis is a powerful technique for generating mutant libraries, and its use has led to the identification of gene functions in various bacterial systems.

Mos1 from Drosophila melanogaster (Robertson and Lampe 1995) and Himar1 from the horn fly Haematobia irritans (Lampe et al. 1996) are among the best-studied mariner transposons. Mos1 is the most frequently used mariner transposon in eukaryotes, while Himar1 has been extensively used for mutagenesis in bacteria (Table 1). A hyperactive mutant of Himar1 was engineered by two amino acid substitutions of the transposase, increasing transposition in Escherichia coli to levels 50 times those of wild type transposase (Lampe et al. 1999). This hyperactive mutant of the Himar1 transposase has been used to generate libraries of mutants in many bacteria (Hava and Camilli 2002; Geoffroy et al. 2003; Hendrixson and DiRita 2004; Stewart et al. 2004; Grant et al. 2005; Jiao et al. 2005; Wu et al. 2006; Liu et al. 2007; Yang et al. 2008; Beare et al. 2009; Murray et al. 2009) and in archae (Zhang et al. 2000).

Table 1 Mariner transposons as tools for mutagenesis in bacteria
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Thousands of random mutants can be readily obtained by transposon mutagenesis, thereby generating extensive libraries of mutants that could be screened for phenotypes affecting diverse aspects of metabolism and physiology, such as amino-acid biosynthesis (Louvel et al. 2005; Rollefson et al. 2009), motility (Hendrixson et al. 2001; Youderian et al. 2003; Braun et al. 2005; Rholl et al. 2008), and biofilm formation (Liberati et al. 2006; Kristich et al. 2008; Rollefson et al. 2009) (Table 1).

In vitro transposition

Transposases mediate efficient Himar1 transposition in vitro and do not require cellular cofactors; thus, this system is particularly well suited to the analysis of naturally competent organisms, such as bacteria belonging to the genera Haemophilus, Streptococcus, Helicobacter, Neisseria, and Campylobacter (Table 1). The in vitro transposition reaction usually consists of target chromosomal DNA, a mariner-derived minitransposon, and purified mariner transposase. The mariner transposon inserts at TA dinucleotides in the target sequence, resulting in a duplication of the TA dinucleotide flanking the insertion. After the repair of single-stranded gaps introduced onto either side of the transposon insertion by the transposase, mutated DNA is introduced into bacteria by natural transformation.

By using a PCR product instead of chromosomal DNA as target DNA, Himar1 in vitro transposition is also suitable for the purpose of target-selected gene inactivation in naturally competent bacteria (Bergé et al. 2001; Guo and Mekalanos 2001).

In vivo transposition

For in vivo transposition, plasmids carrying the mariner-based transposon usually encode a minitransposon that contains a resistance gene, which is flanked by Himar1 inverted repeats. In addition, the plasmid encodes the Himar1 transposase under the control of a strong promoter, optimizing expression of the transposase in transformed bacteria (Rubin et al. 1999). In these studies, a suicide plasmid or a conditional replicative plasmid are used as a delivery vector. Various methods for efficiently delivering mariner elements into bacteria have been described, including electroporation and conjugation (Picardeau 2008). The in vivo transposition system relies on transposase-directed random insertion of a transposon containing an antibiotic resistance gene for positive selection into the host cell. Mariner-based transposons can be efficiently used to generate insertion mutants in bacteria considered to be genetically intractable microorganisms (Bourhy et al. 2005; Braun et al. 2005; Maier et al. 2006).

Determination of transposon insertion sites

DNA sequences that lie adjacent to transposon insertion sites can be characterized using various techniques including plasmid rescue, inverse PCR (Ochman et al. 1988), adaptor-ligation PCR methods (Prod’hom et al. 1998), and semi random PCR (Beeman and Stauth 1997) (Fig. 1). Amplicons or plasmids are then subjected to DNA sequence analysis. In semi-random PCR, a primer of arbitrary sequence is paired with a primer that binds to a region of the transposon. Low annealing temperatures are employed to allow the arbitrary primer to bind at multiple regions in the genome, including those adjacent to the known sequence. A second PCR, in higher stringency conditions, is then carried out with specific primers that bind to the transposon and the 5′ sequence of the arbitrary primers used in the first round of PCR. Ligation-mediated PCR (LMPCR) is another technique for amplifying DNA sequences that flank transposon insertion sites. This method uses one primer that is specific for the transposon and a second that is specific for a synthetic linker ligated to restriction-digested genomic DNA. The transposon carrying a plasmid E. coli origin of replication can be recovered with adjacent stretches of DNA sequences and is then mapped by plasmid rescue. This is achieved by ligation of digested DNA fragments and recovery of replicative plasmid DNA from E. coli transformants. Transposon-chromosomal DNA junction sequences can also be obtained by inverse PCR by ligating digested DNA fragments and priming self-ligation mixtures using transposon-specific primers. Although not widely used, transposon integration sites can also be identified by direct genome sequencing of genomic DNA using a primer within the transposon (Qimron et al. 2003; Murray et al. 2008).

Fig. 1
figure 1

Summary of the basic strategies used for the identification of the transposition insertion sites. a plasmid rescue. b inverse PCR. c Ligation-mediated PCR. d semi random PCR

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Other transposon-based approaches

The use of mariner transposon broadens the repertoire of genetic methods available for the manipulation of bacteria. For example, Himar1 can be used to deliver a FLP recombinase substrate that permits rapid and easy creation of in-frame epitope tag fusions. This system could be employed to analyze protein structure and function (Chiang and Rubin 2002). Transposons such as Himar1 can also deliver gfp or genes encoding other fluorescent proteins to label prokaryotic cells (Bextine et al. 2005).

Transposon-based approaches are powerful tools for the identification of essential and infection-related genes in bacteria. Signature-tagged mutagenesis (STM), which was initially described in Salmonella typhimurium with Tn5 (Hensel et al. 1995), is an in vivo screen approach. This method generates random mutants that contain insertions with various tags to distinguish individual attenuated mutants from a mutant pool. STM has now been developed using Himar1-based vectors to find essential virulence genes in various bacterial hosts (Geoffroy et al. 2003; Grant et al. 2005; Paik et al. 2005).

The use of random mutagenesis in combination with microarray technology has enabled the development of a new method, transposon site hybridization or TraSH, which has the potential to identify genes required for in vitro growth and survival in particular environments. The TraSH assay uses DNA microarrays to determine the locations of transposon insertions in a library of mutants and to compare the effects of each insertion on the representation of clones before and after exposure to a particular environment. Use of Himar1-based TraSH allowed the identification of genes required in vitro in Mycobacterium bovis and Mycobacterium tuberculosis (Sassetti et al. 2001, 2003). The TraSH technique has also allowed the identification of M. tuberculosis genes that are specifically required for survival in mice (Sassetti and Rubin 2003).

Conditionally active genes can be used to examine bacteria during depletion of the essential gene product. TnAraOut is a mariner-based transposon containing an arabinose-inducible promoter. Mutants carrying TnAraOut insertions in front of essential genes are expected to exhibit arabinose dependence and form small colonies on low concentrations of arabinose. This technique has been used to identify essential genes in Vibrio cholerae (Judson and Mekalanos 2000) and Salmonella enteritidis (Kim et al. 2008).

Another technique, called GAMBIT (Genomic analysis and Mapping By In vitro Transposition), provides a powerful system for identifying essential genes in naturally competent bacteria, such as Haemophilus influenzae and Streptococcus pneumoniae. This technique consists of Himar1 in vitro transposition into PCR products, transformation, and genetic footprinting (Akerley et al. 1998).

Conclusion

Transposable elements are ubiquitous components of all living organisms. An important approach for investigating metabolic processes is the generation of large numbers of mutant bacteria by random insertion of transposable elements. As described above, mariner elements are likely to become the favorite genetic tool for assigning functions to the many unannotated genes in genomic databases. Mariner elements have a broad host range, thus making it very likely that these elements are effective for transposon mutagenesis across most bacterial genera.