TnpI recombinase: identification of sites within Tn5401 required for TnpI binding and site-specific recombination

Abstract

The Bacillus thuringiensis class II transposon Tn5401 encodes a recombinase protein, TnpI, that mediates the resolution of cointegrate molecules generated as intermediates during Tn5401 transposition by the TnpA transposase. This recombination event requires a specific target site, or internal resolution site, at which TnpI binds and catalyzes the exchange of DNA strands. Gel mobility shift assays and DNase I footprinting analyses were used to localize the TnpI binding region to the sequence extending from nucleotides 637 to 747 of Tn5401. Deletions within this region blocked TnpI-mediated recombination in vivo. The 12-bp sequence ATGTCC RCTAAY, present in four copies within the TnpI binding region, is proposed to be the recognition sequence for TnpI binding. TnpI also binds to a single copy of this sequence located within the 53-bp terminal inverted repeats of Tn5401. The unique juxtaposition of recombinase and transposase binding sites at the terminal inverted repeats of Tn5401 suggests that TnpI regulates the binding and/or catalytic activity of TnpA transposase.Tn5401 is a class II (15) (Tn3-like) transposable element indigenous to the CryIIIA-producing Bacillus thuringiensis subsp. morrisoni strain EG2158 (3). The 4,837-bp transposon encodes both a transposase protein (TnpA) and a recombinase protein (TnpI) that are required for Tn5401 transposition in B. thuringiensis. The structural organization of Tn5401 resembles that of another B. thuringiensis transposon, Tn4430, but the two transposons are only distantly related as judged by nucleotide and amino acid sequence identity (3).The TnpI recombinase, related to the phage integrase family of site-specific recombinases, catalyzes a recombination event that resolves cointegrate molecules formed as intermediates during Tn5401 transposition. Previously, it was shown that a mutation in the tnpI gene of the Tn5401-based transposon vector pEG922 leads to the accumulation of cointegrate molecules in B. thuringiensis (3), as would be predicted from the Tn3 model of replicative transposition (9,22). This recombination event presumably occurs at a specific site, referred to as an internal resolution site (IRS).Sequence analysis of Tn5401 has revealed a likely candidate for the IRS (3). A 12-bp sequence element, ATGTCCRCT AAY, occurs four times in the intergenic region between the orf1 coding region and the tnpI gene (Fig. 1), two copies of which form a 28-bp imperfect inverted repeat (IR). A similar sequence arrangement, albeit a different conserved sequence, is found in the tnpI promoter region of Tn4430 (17) as well as in the target sites of other site-specific recombinases of the integrase-like recombinase family (see reference 23 and references therein). In Tn5401, direct repeats of the 12-bp sequence are also positioned between the Ϫ10 and Ϫ35 sites of the divergent promoters for orf1 and tnpI (Fig. 1), an observation consistent with studies implicating TnpI as a negative regulator of transcription from these promoters (3).In addition, one copy of the conserved se...

Characterization of XerC‐ and XerD‐dependent CTX phage integration in Vibrio cholerae

“…For example, resolution of the E. coli F plasmid requires a 200 bp sequence that contains two discrete binding sites for the F-encoded ResD tyrosine recombinase (reviewed in Hallet et al, 2004). Likewise, the tyrosine recombinases involved in integron gene capture and in resolution of Tn4430 and Tn5401 bind to additional accessory sites adjacent to the recombination core sites (Mahillon and Lereclus, 1988;Baum, 1995;Hansson et al, 1997;Gravel et al, 1998;Partridge et al, 2000;Recchia and Sherratt, 2002). The role of these auxiliary sites in recombination has not been defined.…”

Self‐control in DNA site‐specific recombination mediated by the tyrosine recombinase TnpI

“…The four-motif structure of Tn4430 IRS is conserved in Tn5401, another B. thuringiensis transposon that encodes a TnpI recombinase (Baum, 1995), suggesting that both transposons use the same mechanism to control cointegrate resolution. Intriguingly, a similar organization of the recombination site was recently reported for the attI integration site of integrons, whereas the attC site carried by the mobile gene cassettes exhibits a different structure containing two pairs of inversely oriented binding motifs for the IntI recombinase (Collis et al, 1998;Gravel et al, 1998;Partridge et al, 2000;Recchia and Sherratt, 2002).…”

Comparison of the structure–activity relationships of the integron-associated recombination sites attI3 and attI1 reveals common features

“…A similar arrangement of four integrase-binding sites, a simple site and two directly oriented sites, has also been found in the site recognized by the TnpI tyrosine recombinase of Tn5401 which is responsible for resolution of cointegrates formed during transposition (Baum, 1995). However, the Tn5401 resolution site is longer than the attI sites and the position of the directly oriented accessory sites with respect to the simple site is not the same (Fig.…”