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Journal of Bacteriology, May 2007, p. 3918-3921, Vol. 189, No. 10
0021-9193/07/$08.00+0     doi:10.1128/JB.01863-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Target Site Selection of Pseudomonas putida Transposon Tn4652{triangledown} ,{dagger}

Paula Ann Kivistik, Maia Kivisaar, and Rita Hõrak*

Estonian Biocentre and Institute of Molecular and Cell Biology, Tartu University, 51010 Tartu, Estonia

Received 12 December 2006/ Accepted 2 March 2007


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ABSTRACT
 
We analyzed the target preferences of a Tn3 family transposon Tn4652. Alignment of 93 different insertion sites revealed a consensus sequence which resembles that of Tn3, indicating that despite a low similarity between Tn4652 and Tn3 transposases, their target site recognition is conserved.


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TEXT
 
Transposons insert into various sites in the host genome while most of them still display some degree of target site selectivity. Mobile elements recognize a particular target site according to the DNA sequence and structure (7, 11, 14, 27). The degree of DNA supercoiling (21), the level of transcription (5, 8, 33), and ongoing replication (25) may also influence attractiveness of the target. The target site can even determine initiation of transposition. Namely, excision of Tn7 occurs only after the target region attTn7 is found in the Escherichia coli chromosome (2).

Pseudomonas putida transposon Tn4652 is a 17-kb-long deletion derivative of the toluene degradation transposon Tn4651 (32). A remarkable feature of Tn4652 is its activation under stress conditions due to involvement of two stationary growth phase proteins, {sigma}S and integration host factor, in its regulation (13, 15, 16). Transposition of native Tn4652 has been examined in a starvation assay selecting for insertions of chromosomally located Tn4652 into plasmid pEST1332 in front of the promoterless phenol monooxygenase gene pheA (15). Translocation of Tn4652 activates the pheA gene due to generation of a fusion promoter at the insertion site, resulting in accumulation of phenol-utilizing Phe+ mutants on phenol minimal plates (22). Fusion promoters between the transposon and the target DNA may be created by both ends of Tn4652, and both types of promoters are equally strong (31). Surprisingly, Tn4652 exhibits a strong orientation bias upon insertion, with the majority of insertions occurring with the right end of Tn4652 toward the pheA (17, 22). It has been shown that orientation of the transposon Tn7 in E. coli chromosome correlates with the direction of replication (25, 26). Therefore, we wondered whether the direction of replication through the pheBA target region could affect orientation of Tn4652. In addition, as the starvation assay allowed identification of only few target sites, a mating-out assay was developed to characterize the targeting preferences of Tn4652 more precisely.

Orientation bias of Tn4652 relative to the pheBA target region is maintained upon inversion of the target region on the plasmid. To test the effect of the direction of plasmid replication on Tn4652 orientation, the pheBA operon-containing region in plasmid pEST1332 was inverted, resulting in a new target plasmid pEST2331 (Table 1). The starvation assay was performed on P. putida PaW85 wild-type strain carrying either pEST1332 or pEST2331. Bacteria were grown overnight in LB medium at 30°C. Approximately 1 x 108 cells from five independent P. putida cultures containing target plasmids were plated onto phenol minimal plates, and accumulation of mutant Phe+ colonies was monitored upon incubation of plates at 30°C for 7 days. Both target plasmids enabled a similar frequency of generation of Phe+ colonies (Fig. 1). Analysis of Tn4652 insertions in target plasmids by PCR (see Table S1 in the supplemental material) demonstrated that over 95% of Phe+ mutants, in the case of pEST2331, arose due to transposition of Tn4652, which is in accordance with our previous results obtained with pEST1332 (15). Thus, inversion of the pheBA operon did not affect its attractiveness as a target for Tn4652. The analysis also demonstrated that orientation of Tn4652 with respect to pheA gene was retained: in most cases the right end of the transposon was oriented toward the pheA in either target plasmid (Table 2). However, as the target region of Tn4652 is inversed in pEST2331, orientation bias of Tn4652 became switched relative to the origin of replication. This suggests that the orientation bias of Tn4652 observed in a starvation assay is not related to the direction of plasmid replication, but rather, the right end of the transposon is preferred in fusion promoter creation.


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  		TABLE 1. Bacterial strains and plasmids


Figure 1
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  		FIG. 1. Accumulation of Phe+ colonies due to transposition of Tn4652 into target plasmids pEST1332 (Tn4652->pEST1332) and pEST2331 (Tn4652->pEST2331). Results of five independent experiments (mean ± standard deviation) are presented.


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  		TABLE 2. Insertions of native Tn4652 in target plasmids pEST1332 and pEST2331

Tn4652 inserts randomly around target plasmids in mating-out assay. The starvation assay allows the detection of Tn4652 insertions in a few specific positions that serve as {sigma}70 promoter –10 hexamers in the creation of fusion promoters alongside a –35 hexamer within the transposon end. To expand the range of Tn4652 targets, a mating-out assay was developed. For that, the chromosomally located Tn4652 was tagged with the kanamycin resistance gene, creating a transposon donor strain P. putida PaW4652Km (Table 1). Different plasmids (Fig. 2) serving as targets for Tn4652Km were introduced into PaW4652Km. Plasmids pEST1332 and pEST2331 were employed to investigate whether Tn4652 will insert into the same sites as in the starvation assay. Plasmid pAYC32, lacking the pheBA DNA, was used as a reference. In addition to RSF1010-based plasmids pEST1332, pEST2331, and pAYC32, an RK2-based plasmid pPR9TT was used to study target preference of Tn4652 in a dissimilar replicon. Tn4652 insertions into target plasmids were examined after mating the donor strain PaW4652Km with a recipient P. putida PaW85tet (Table 1). Mixtures of donor and recipient cultures were spotted onto an LB plate and incubated overnight at 30°C, and dilutions of each mating mixture were plated onto selective media. Transconjugants appearing on kanamycin-carbenicillin-tetracycline-selective plates were analyzed by PCR to detect Tn4652 insertions in target plasmids (see Table S1 in the supplemental material).


Figure 2
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  		FIG. 2. Maps of target plasmids of Tn4652. Tn4652 insertion sites detected in mating-out assay are marked with dashes, with the outer dash representing counterclockwise orientation of Tn4652 (demonstrated in the case of pPR9TT) and the inner dash representing clockwise orientation. Identical insertion sites are highlighted with black dots. IS1411 and different genes are marked as gray boxes, and the direction of transcription is shown by arrow. Origins of plasmid replication and transfer are marked as white boxes. Restriction sites PvuII (P) and Ecl136II (E) used to extract and reverse the pheBA operon are denoted on pEST1332. Plasmid pEST2331 was analyzed only for Tn4652 insertions in pheBA region.

Tn4652 insertions were distributed quite evenly over target plasmids (Fig. 2), suggesting that Tn4652 has no preferred target region. Moreover, most of the insertion sites detected were unique, and only three sites in RSF1010 plasmids were used more often than once (Fig. 2). Interestingly, none of the insertion sites in pEST1332 and pEST2331 coincided with those of a starvation assay. Tn4652 insertions in target plasmids revealed a slight orientation bias: about 70% of insertions occurred in one orientation (Fig. 2). This suggests that some plasmid-related feature may have a subtle impact on the orientation of Tn4652 upon insertion.

Identification of the Tn4652 target consensus sequence. To analyze Tn4652 target site specificity more rigorously, 93 different Tn4652 target sites were aligned (Fig. 3). The compared region included a 5-bp-long direct repeat characteristic of Tn4652 and 10-bp-long sequences at both sides of the direct repeat. First, a sequence logo was generated (Fig. 3A) using WebLogo analysis (http://weblogo.berkeley.edu), indicating that most conserved positions in Tn4652 target are dr2 and dr4 of the direct repeat and ±1 of the flanking region. Probability calculations for each position accounting for the average G/C content of target plasmids (Fig. 3B) suggest that up to 19 positions of 25 analyzed may be considered statistically conserved, as distribution of nucleotides in these positions differed significantly from the random (P < 0.05). The preferred nucleotide pattern was prominent in target duplication, revealing an A/T-rich conserved sequence T(A/T)(T/A)(T/A)(A/T). In accordance with WebLogo results, the highest conservation in the direct repeat was observed for positions dr2 and dr4. Flanking positions ±1, ±2, and ±5 also demonstrated a highly biased nucleotide distribution (Fig. 3B) (P < 0.001). Notably, in contrast to the A/T-rich direct repeat, the bordering positions are mostly occupied by G or C nucleotides. There is particularly strong selection against A or T at positions ±1. This characteristic may be important for the target site selection, as the junction between the direct repeat and the flanking sequence is the site cleaved by the transposase. One may hypothesize that an A/T-rich sequence surrounded by a G/C-rich region has higher deformability and is thereby more easily attacked by the transposase bound to this region. For example, DNA bending has been reported to affect the target site selection by some mobile elements (1, 20, 27).


Figure 3
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  		FIG. 3. Tn4652 target site consensus sequence. (A) Sequence logo drawn from 93 distinct Tn4652 insertion sites. Positions dr1 to dr5 represent nucleotides in the direct repeat (dr) generated by Tn4652 transposition, –1 to –10 are nucleotides adjacent to the right end of Tn4652, and positions 1 to 10 are those next to the left end of Tn4652. The degree of sequence conservation at each position is indicated as a total height of a stack of letters, measured in arbitrary "bit" units, with two bits possible at each position. (B) Matrix describing Tn4652 insertion site specificity. A plus sign (+) designates positions which demonstrated nonrandom distribution of nucleotides according to the {chi}2 test; P values of less than 0.05, 0.01, and 0.001 are presented. Conserved nucleotides are indicated underneath. Pu, purine; Py, pyrimidine.

In addition to observed nucleotide conservation, the target positions dr2/dr4 and ±1 of Tn4652 target site tend to be palindromic according to the {chi}2 test (P = 0.002 for both position pairs). Furthermore, 11 insertion sites of 93 revealed palindromy at both dr2/dr4 and ±1 positions (P = 0.026). This suggests that the transposition complex of Tn4652 makes symmetrical contacts with its target. Notably, the target sequences of several other mobile elements, like those of IS10, Tn3, Mu, and IS30 (4, 7, 11, 23), are often symmetrical.

Comparison of Tn4652 and Tn3 consensus sequences. Tn4652 is a distantly related member of Tn3 family according to transposase gene alignment (13). Though the Tn4652 and Tn3 transposases show only about 30% homology (13), their target selection criteria are remarkably similar. Tn3 preferably inserts into a 5-bp TA(T/A)TA sequence (7, 19) which clearly resembles the Tn4652 consensus T(A/T)(T/A)(T/A)(A/T). Furthermore, the A/T-rich central consensus regions of both Tn4652 and Tn3 are flanked by G/C-rich sequences (7, 19). However, more detailed comparison reveals some discrepancies between the insertion sites preferred by Tn4652 and Tn3. The most important positions in Tn3 target duplication are dr1 and dr5 (19) versus dr2 and dr4 in Tn4652 target duplication. Additionally, while flanking positions ±1, ±2, and ±5 are highly conserved in the target of Tn4652, the positions +3 and –3 are most significant in the Tn3 consensus (19, 29). Nevertheless, the overall target recognition of Tn4652 and Tn3 is certainly similar, raising an interesting question of whether the target site selection criteria of other Tn3 family transposons resemble those of Tn4652 and Tn3.


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ACKNOWLEDGMENTS
 
We are grateful to Tiina Alamäe, Inga Sarand, Heili Ilves, and Marta Putrins for critically reading the manuscript.

This work was supported by grant 5758 from the Estonian Science Foundation and by grant HHMI 55000316 from the Howard Hughes Medical Institute International Research Scholars Program to M.K. and by grant 6025 from the Estonian Science Foundation to R.H.


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FOOTNOTES
 
* Corresponding author. Mailing address: Estonian Biocentre and Institute of Molecular and Cell Biology, Tartu University, 23 Riia Street, 51010, Tartu, Estonia. Phone: 372 7 375015. Fax: 372 7 420286. E-mail: rhorak{at}ebc.ee Back

{triangledown} Published ahead of print on 9 March 2007. Back

{dagger} Supplemental material for this article may be found at http://jb.asm.org/. Back


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Journal of Bacteriology, May 2007, p. 3918-3921, Vol. 189, No. 10
0021-9193/07/$08.00+0     doi:10.1128/JB.01863-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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