IS903 transposase mutants that suppress defective inverted repeats

The inverted repeats (IRs) of the insertion element IS903 are composed of two functional regions. An inner region, consisting of basepairs 6-18, is the transposase binding site. The outer region (positions 1-3) is not contacted during initial transposase binding, but is essential for efficient transposition. We have examined the interaction of the IR with the transposase by isolating transposase suppressors of IR mutations. These suppressors define two patches within the N-terminus of the protein. One class of suppressors, which rescued the majority of outer IR mutants tested, contained mutations in close proximity to an aspartate residue (D121) believed to form part of the catalytic DDE motif, suggesting that their suppressive effect is in the positioning of the catalytic site at the terminus of the transposon. The hypertransposition phenotype of mutant VA119 is also consistent with this hypothesis. The second class was more allele specific and preferentially suppressed a mutation at position 3 of the IR. Finally, we showed that mutations at the termini of the IR elevate the frequency of cointegrate formation by IS903. Other outer IR mutations did not have this effect. These data are consistent with the terminal bases of the transposon playing multiple and distinct roles in transposition.     

The regulatory role of the IS 1-encoded InsA protein in transposition

We show here that the protein InsA, which is encoded by IS 1 and binds specifically to the terminal inverted repeats of this insertion sequence, negatively regulates IS 1 transposition activity. We demonstrate that it inhibits both IS 1-mediated cointegrate formation and transposition of a synthetic IS 1-based transposon (‘omegon’Ω-on). These results also indicate that the Ω-on which does not itself encode IS 1 transposition functions can be complemented in trans, presumably by the copies of IS 1 resident in the Escherichia coli chromosome. Using insA-lacZ gene fusions, we show that at least part of this effect can be explained by the ability of InsA to repress expression of IS 1-encoded genes both in cis or in trans. The experiments involving Ω-on transposition raise the possibility that InsA inhibits transposition directly by competition with the transposase for their cognate site within the ends of IS 1.     

Direct selection of IS903 transposon insertions by use of a broad-host-range vector: isolation of catalase-deficient mutants of Actinobacillus actinomycetemcomitans

Transposon mutagenesis in bacteria generally requires efficient delivery of a transposon suicide vector to allow the selection of relatively infrequent transposition events. We have developed an IS903-based transposon mutagenesis system for diverse gram-negative bacteria that is not limited by transfer efficiency. The transposon, IS903phikan, carries a cryptic kan gene, which can be expressed only after successful transposition. This allows the stable introduction of the transposon delivery vector into the host. Generation of insertion mutants is then limited only by the frequency of transposition. IS903phikan was placed on an IncQ plasmid vector with the transposase gene located outside the transposon and expressed from isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible promoters. After transposase induction, IS903phikan insertion mutants were readily selected in Escherichia coli by their resistance to kanamycin. We used IS903phikan to isolate three catalase-deficient mutants of the periodontal pathogen Actinobacillus actinomycetemcomitans from a library of random insertions. The mutants display increased sensitivity to hydrogen peroxide, and all have IS903phikan insertions within an open reading frame whose predicted product is closely related to other bacterial catalases. Nucleotide sequence analysis of the catalase gene (designated katA) and flanking intergenic regions also revealed several occurrences of an 11-bp sequence that is closely related to the core DNA uptake signal sequence for natural transformation of Haemophilus influenzae. Our results demonstrate the utility of the IS903phikan mutagenesis system for the study of A. actinomycetemcomitans. Because IS903phikan is carried on a mobilizable, broad-host-range IncQ plasmid, this system is potentially useful in a variety of bacterial species.     

Intramolecular transposition of insertion sequence IS91 results in second-site simple insertions

A series of plasmids carrying an IRL-kan-IRR transposable cassette, in which IRL and IRR are the left- and right-terminal sequences of IS91, have been constructed. These cassettes could be complemented for transposition with similar efficiency when IS91 transposase was provided either in cis or in trans. A total of 87% of IS91 transposition products were simple insertions of the element, while the remaining 13% were plasmid fusions and co-integrates. When transposase expression was induced from an upstream lac promoter, transposition frequency increased approximately 100-fold. An open reading frame (ORF) present upstream of the transposase gene, ORF121, could be involved in target selection, as mutations affecting this ORF were altered in their insertion specificity. Intramolecular rearrangements were analysed by looking at transposition events disrupting a chloramphenicol resistance gene (cat ) located outside the transposable cassette. Plasmid instability resulting from insertion of an extra copy of IRL-kan-IRR within the cat gene was observed; transposition products contained a second copy of the cassette inserted either as a direct or as an inverted repeat. No deletion or inversion of the intervening DNA was observed. These results could be explained as a consequence of intramolecular transposition of IS91 according to a model of rolling-circle transposition.     

IS10 transposase mutations that specifically alter target site recognition.

IS10 inserts preferentially into particular hotspots. We describe here mutations of IS10 transposase, called 'ATS' that confer Altered Target Specificity. These mutations yield a general relaxation in target specificity but do not affect other aspects of transposition. Thus, the preference for specific nucleotide sequences at the target site can be cleanly separated from other steps of the transposition reaction. Eleven ATS mutations identified in a genetic screen occur at only two codons in transposase, one in each of two regions of the protein previously implicated in target site interactions (Patch I and Patch II). Genetic analysis suggests that mutations at the two ATS codons affect the same specific function of transposase, thus raising the possibility that Patch I and Patch II interact. For wild-type IS10, insertion specificity is determined in part by a specific 6 bp consensus sequence and in part by the immediately adjacent sequence context of the target DNA. The ATS mutations do not qualitatively alter the hierarchy with which base pairs are recognized in the consensus sequence; instead, sites selected by ATS transposase exhibit a reduction in the degree to which certain base pairs are preferred over others. Models for the basis of this phenotype are discussed.     

Orientation of IS50 transposase gene and IS50 transposition.

Reversal of transposase gene orientation with respect to the nonidentical ends of IS50 strongly decreased IS50 transposition in both Dam- and Dam+ hosts. In either orientation, IS50 transposase expression was unaffected. These effects were independent of the surrounding DNA context. This shows that the efficiency of IS50 transposition is dependent on transposase gene orientation. The transposition frequencies of transposons utilizing inverted IS50 inside ends (IE), IE-IE transposons, were lower than either outside end (OE)-IE or OE-OE transposons.     

Characterization of functionally important sites in the bacteriophage Mu transposase protein

The 663 amino acid Mu transposase protein is absolutely required for Mu DNA transposition. Mutant proteins were constructed in vitro in order to locate regions of transposase that may be important for the catalysis of DNA transposition. Deletions in the A gene, which encodes the transposase, yielded two stable mutant proteins that aid in defining the end-specific DNA-binding domain. Linker insertion mutagenesis at eight sites in the Mu A gene generated two proteins, FF6 and FF14 (resulting from two and four amino acid insertions, respectively, at position 408), which were thermolabile for DNA binding in vitro at 43°C. However, transposition activity in vivo was severely reduced for all mutant proteins at 37°C, except those with insertions at positions 328 and 624. In addition, site-specific mutagenesis was performed to alter tyrosine 414, which is situated in a region that displays amino acid homology to the active sites of a number of nicking/closing enzymes. Tyrosine 414 may reside within an important, yet non-essential, site of transposase, as an aspartate-substituted protein had a drastically reduced frequency of transposition, while the remaining mutants yielded reduced, but substantial, frequencies of Mu transposition in vivo.     

Tipping the balance between replicative and simple transposition

The bacterial insertion sequence IS903 has the unusual ability to transpose both replicatively and non-replicatively. The majority of products are simple insertions, while co-integrates, the product of replicative transposition, occur at a low frequency (<0.1% of simple insertions). In order to define the critical steps that determine the outcome of IS903 transposition, we have isolated mutants that specifically increase the rate of replicative transposition. Here we show that the nucleotide immediately flanking the transposon influences both overall transposition frequency and co-integrate formation. In particular, when the 3'-flanking nucleotide is A, co-integrates are increased 500-fold compared with a 3' C. In addition, we have isolated five transposase mutants that increase replicative transposition. These residues are close to the catalytic residues and are thus likely to be part of the active site. These are the first transposase mutations described that affect the product of transposition. Our results are consistent with the hypothesis that a delay in cleavage of the 5'-flanking DNA will increase the effective half-life of the 3'-nicked transposon intermediate and consequently enhance co-integrate formation.     

DNA-binding activity and subunit interaction of the mariner transposase

Mos1 is a member of the mariner/Tc1 family of transposable elements originally identified in Drosophila mauritiana. It has 28 bp terminal inverted repeats and like other elements of this type it transposes by a cut and paste mechanism, inserts at TA dinucleotides and codes for a transposase. This is the only protein required for transposition in vitro. We have investigated the DNA binding properties of Mos1 transposase and the role of transposase–transposase interactions in transposition. Purified transposase recognises the terminal inverted repeats of Mos1 due to a DNA-binding domain in the N-terminal 120 amino acids. This requires a putative helix–turn–helix motif between residues 88 and 108. Binding is preferentially to the right hand end, which differs at four positions from the repeat at the left end. Cleavage of Mos1 by transposase is also preferentially at the right hand end. Wild-type transposase monomers interact with each other in a yeast two-hybrid assay and we have used this to isolate mutations resulting in reduced interaction. These mutations lie along the length of the protein, indicating that transposase–transposase interactions are not due to a single interaction domain. One such mutation which retains both DNA-binding and catalytic activity has greatly reduced ability to excise Mos1 from plasmid DNA through coordinate cleavage of the two ends and transposition in vitro is lowered to a level 20-fold below that of the wild-type. This suggests that transposase–transposase interaction is required to form a synaptic complex necessary for coordinate cleavage at the ends of Mos1 during transposition. This mutant enzyme allows insertion at dinucleotides other than TA, including sequences with GC base pairs. This is the first example of a mariner/Tc1 transposase with altered target specificity.     

Binding of the IS903 transposase to its inverted repeat in vitro.

We have purified the transposase of IS903 in three different ways. We find that transposase expressed as a fusion protein with either glutathione-S-transferase or maltose-binding protein is soluble and can be purified rapidly using affinity chromatography. The third purification requires extracting the native transposase from an insoluble pellet using an alkaline pH buffer. All three proteins bind specifically to the ends of IS903 and give identical patterns of protection when challenged with DNase I. We have used the more stable fusion proteins to examine transposase--DNA interactions in vitro. Methylation interference experiments have identified critical bases for transposase binding; methylated purines that inhibit binding all lie within the inner part of the 18 bp inverted repeat (bp 7-16). Moreover, the positions and identities of these purines suggest that the transposase interacts with base pairs in adjacent major and minor grooves. Binding assays with mutant inverted repeats confirm that transposase binding is sensitive to sequence changes only within this inner region. We propose that the transposase binding site is limited to this domain of the inverted repeat. These data are consistent with our previous analysis of the behaviour of mutant ends in vivo, from which we postulated that the inverted repeat was composed of two functional domains; an inner binding domain (bp 6-18), which included a region of minor groove interactions, and an outer domain that was involved in a step subsequent to transposase binding.     

Carboxyl-terminal mutants of phage Mu transposase

A study of the properties of deletion mutants at the 3’ end ofA, the gene encoding the transposase protein of phage Mu, shows that the mutants are defective in the high-frequency non-replicative transposition observed early after Mu infection as well as the high-frequency replicative transposition observed during Mu lytic growth. They show near-normal levels of lysogenization, low frequency transposition and precise excision. The mutants behave as if they are “blind” to the presence of Mu B, a protein whose function is essential for the high frequency of both replicative and non-replicative Mudna transposition. We have sequenced these deletion mutants as well as the amber mutant A 7110 which is known to be defective in replicative transposition.A 7110 maps at the 3’ end of geneA. We suggest that the carboxyl-terminal region of the A-protein is involved in protein-protein interactions, especially with the B-protein. We also show in this study that mutations upstream of the Shine-Dalgarno sequence of geneA and within the preceding genener, perturb the synthesis of A-protein and that higher levels of A-protein cause an inhibition ofA activity.     

Ds transposition mediated by transient transposase expression in Heiracium aurantiacum

The feasibility of using transient transposase expression to mobilize Ds elements for gene tagging in Hieracium aurantiacum was evaluated. A T-DNA construct carrying the Ac transposase gene and either a visible marker gene (uidA) or the conditionally-lethal marker gene (codA) was transferred to H. aurantiacum leaf discs (previously transformed with a Ds element) by co-cultivation with Agrobacterium tumefaciens. Shoots were regenerated directly from the co-cultivated leaf discs under selection for antibiotic resistance resulting from Ds excision. Most regenerants carried unique transposition events. Of 84 regenerated plants, twenty one (25%) did not express the marker gene and the DNA coding sequence of the transposase could not be detected in seven (8.3%). Potential advantages of this method over conventional gene-tagging methods are: rapid recovery of individual transposition events; regenerated plants are isogenic; and the transient nature of transposase expression should facilitate the stabilisation of the transposed element.     

Truncated forms of IS911 transposase downregulate transposition

IS911 naturally produces transposase (OrfAB) derivatives truncated at the C-terminal end (OrfAB-CTF) and devoid of the catalytic domain. A majority species, OrfAB*, was produced at higher levels at 42 degrees C than at 30 degrees C suggesting that it is at least partly responsible for the innate reduction in IS911 transposition activity at higher temperatures. An engineered equivalent of similar length, OrfAB[1-149], inhibited transposition activity in vivo or in vitro when produced along with full-length transposase. We isolated several point mutants showing higher activity than the wild-type IS911 at 42 degrees C. These fall into two regions of the transposase. One, located in the N-terminal segment of OrfAB, lies between or within two regions involved in protein multimerization. The other is located within the C-terminal catalytic domain. The N-terminal mutations resulted in reduced levels of OrfAB* while the C-terminal mutation alone appeared not to affect OrfAB* levels. Combination of N- and C-terminal mutations greatly reduced OrfAB* levels and transposition was concomitantly high even at 42 degrees C. The mechanism by which truncated transposase species are generated and how they intervene to reduce transposition activity is discussed. While transposition activity of these multiply mutated derivatives in vivo was resistant to temperature, the purified OrfAB derivatives retained an inherent temperature-sensitive phenotype in vitro. This clearly demonstrates that temperature sensitivity of IS911 transposition is a complex phenomenon with several mechanistic components. These results have important implications for the several other transposons and insertion sequences whose transposition has also been shown to be temperature-sensitive.     

The N-terminus of Himar1 mariner transposase mediates multiple activities during transposition

Mariner family transposons are perhaps the most widespread transposable elements of eukaryotes. While we are beginning to understand the precise mechanism of transposition of these elements, the structure of their transposases are still poorly understood. We undertook an extensive mutagenesis of the N-terminal third of the transposase of the Himar1 mariner transposon to begin the process of determining the structure and evolution of mariner transposases. N and C-terminal deletion analyses localized the DNA binding domain of Himar1 transposase to the first 115 amino acids. Alanine scanning of 23 selected sites within this region uncovered mutations that not only affected DNA binding but DNA cleavage as well. The behavior of other mutations strongly suggested that the N-terminus is also involved in multimerization of the transposase on a single inverted terminal repeat and in paired ends complex formation which brings together the two ends of the transposon. Finally, two hyperactive mutations at conserved sites suggest that mariner transposases are under a pattern of stabilizing selection in nature with regard to how efficiently they mediate transposition, resulting in a population of “average” transposons.     

Regulation of IS1 transposition by the insA gene product

The IS1 element contains two adjacent genes called insA and insB, both required for IS1 transposition and IS1-mediated plasmid cointegration. These two genes are transcribed polycistronically from the promoter in the left terminal inverted repeat of IS1 (insL). We constructed overexpression systems of these genes with the tac promoter, which are regulated by an exogenous inducer, isopropyl-beta-D-thiogalactopyranoside (IPTG). Then we have examined, under various conditions of induction with IPTG, how overexpression of these genes affects IS1 transposition, using an assay based on plasmid cointegration. When the insA and insB genes were organized identically to the wild-type IS1 genes and simultaneously expressed using low concentrations of IPTG, activity of a mutant IS1 in cis was restored, but not in trans. Higher IPTG concentrations resulted in lower transposition activity. Expression in trans of insA and insB results in a 50 to 100-fold reduction of the frequency of cointegration mediated by wild-type IS1. Such a reduction is also observed when only the insA gene is overexpressed in trans. Overexpression of either mutant insA or insB does not affect the cointegration event. Tests with the insA-lacZ fusion gene showed that the InsA product inhibits the expression of IS1 genes directed by its own promoter in insL. These results suggest that the InsA product regulates IS1 transposition by inhibiting expression of IS1 transposition genes in addition to acting as part of a transposase complex.