Site-specific recombination system encoded by toluene catabolic transposon Tn4651

Abstract

The 56-kb class II toluene catabolic transposon Tn4651 from Pseudomonas putida plasmid pWW0 is unique in that (i) its efficient resolution requires, in addition to the 0.2-kb resolution (res) site, the two gene products TnpS and TnpT and (ii) the 2.4-kb tnpT-res-tnpS region is 48 kb apart from the tnpA gene (M. Tsuda, K.-I. Minegishi, and T. Iino, J. Bacteriol. 171:1386-1393, 1989). Detailed analysis of the 2.4-kb region revealed that the tnpS and tnpT genes encoding the putative 323- and 332-amino-acid proteins, respectively, were transcribed divergently with an overlapping 59-bp sequence in the 203-bp res site. The motifs (the R-H-R-Y tetrad in domains I and II with proper spacing) commonly conserved in the integrase family of site-specific recombinases were found in TnpS. In contrast, TnpT did not show any significant amino acid sequence homology to the other proteins that are directly or indirectly involved in recombination. Analysis of site-specific recombination under the Escherichia coli recA cells indicated that (i) the site-specific resolution between the two copies of the res site on a single molecule was catalyzed by TnpS, (ii) the functional res site was located within a 95-bp segment, and (iii) TnpT appeared to have the role of enhancing the site-specific resolution. It was also found that TnpS catalyzed the site-specific recombination between the res sites located at two different molecules to form a cointegrate molecule. Site-specific mutagenesis of the conserved tyrosine residue in TnpS led to the loss of both the resolution and the integration activities, indicating that such a residue took part in both types of recombination.

FIG. 1.

Structure of Tn4651. Restriction site abbreviations: A, AatII; Ap, ApaI; B, BamHI; E, EcoRI; H, HindIII; K, KpnI; N, NcoI; Sa, SacI; Sb, SacII; Sm, SmaI; St, StuI; and X, XbaI. Restriction sites derived from the vectors are in parentheses. (A) Schematic structures of Tn4651 (13, 35) and the related transposon Tn5041 (17). The following abbreviations and symbols are used: xyl, toluene-degrading genes; U, upper pathway operon; M, meta pathway operon; mer, genes for resistance to mercuric ion; TnpC, a regulator that controls negatively the TnpA expression at the posttranscriptional level (13); filled arrowhead, IR; and open arrowhead, IS1246 (26). The arrow indicates the transcriptional direction of the genes and operons. (B) Detailed structure of the tnpS-res-tnpT region. The numbers above the restriction sites are the base positions obtained by defining the upstream XbaI site as position 1. The short arrow below the map is the sequence in the primer used for amplification of DNA fragments or for primer extension analysis. Note that the tnpS and tnpT genes had been indicated to be located within the XbaI-SacII and AatII-SmaI fragments, respectively (34). (C) Structures of the pUC- or pBR322-based plasmids carrying tnpS, tnpT, and both genes. The detailed procedures for construction of these plasmids are described in Materials and Methods. The size of the Km^r gene on pGEN7K is arbitrary. While pGEN59 and pGEN77 carry the wild-type tnpS gene, pGEN95 and pGEN96 carry the Y293F mutation (triangle) in the tnpS gene.

FIG. 2.

Construction of pMT299 and R388 derivatives carrying various parts of the res site. Only the relevant restriction sites are depicted, and their abbreviations are as described in the legend of Fig. 1 except for the following: Hc, HincII; P, PstI; and Sc, ScaI. The filled arrow indicates the res site. The white arrowheads above some plasmids indicate the primers used for amplification of parts of the res sites. The PCR product with primers M4 and RES1 was used to obtain res1 present on pGENKres1, pMT299res1, pGEN101, and pGEN388; that with M4 and RES2 was used to obtain res2 on pGENKres2, pMT299res2, pGEN102, and pGEN389; and that with M4 and RES3 was used to obtain res3 on pGENKres3, pMT299res3, and pGEN103. For construction of pGENres6, PCR was carried out by using pMT299res2 as the template with primers RES6 and 184E. See Fig. 3 for details. The bold line indicates the pMT299-derived region. The Km^r gene in all of the plasmids is transcribed in a leftward direction.

FIG. 3.

Structure of the Tn4651 res site. (A) Sequence of the res site. The numbers at the left are the base positions obtained by taking the upstream XbaI site (Fig. 1B) as position 1. The transcription start sites of tnpS and tnpT are indicated by closed circles. The putative −35 and −10 sequences for tnpS and tnpT and the putative SD (S.D.) sequence for tnpT are boxed, and the predicted translation start sites of the tnpS and tnpT genes are shown by bold arrows. The 9-bp IRs are indicated by a pair of thin arrows, and the putative IHF-binding site (9) is doubly overlined. The oligonucleotides for PCR amplification of various parts of the res site are indicated as white boxes above or below the sequence. (B) Summary of the Tn4651 site-specific recombination analysis. The structure of one of the two copies of the res region on each plasmid is schematically drawn. The number above the structure indicates the nucleotide position (Fig. 1B). Open and filled arrows indicate the 9-bp IRs and the putative IHF binding site, respectively. Ssr, detection of site-specific resolution of the plasmid on the agar plates; PCR, detection of the excised and nonreplicating DNA by PCR with primers 4Km1 and 4Km2 (see Fig. 6); Ssi, detection of site-specific integration between the res sites located at two different DNA molecules (Table 2); +, detected; −, not detected.

FIG. 4.

Comparison of TnpS with other members in the integrase family of site-specific recombinases. The consensus sequences in this family are depicted at the top of the figure; the invariant R-H-R-Y tetrad residues in the family are indicated by the capital and boldface letters, and the highly conserved amino acid residue is indicated by the small letter (23). The number beside the amino acid sequence is the amino acid position. The mutagenized tyrosine residue in TnpS is indicated by an arrowhead. The accession numbers of the nucleotide sequences for the proteins are as follows: Tn4651 TnpS (AB077820); pADP-1 Int, putative integrase from plasmid pADP-1 (U66917); Coxiella Int, integrase-like protein from C. burnetii (Y15898); and Tn5041 OrfI, integrase-like protein from Tn5041 (X98999).

FIG. 5.

Resolution and cointegration of plasmids carrying Tn4651 res site. (A) Resolution of plasmid that carried the Km^r gene flanked by two directly repeated copies of the res site. The JM109 derivative carrying the substrate and helper plasmids was cultivated for 48 h in LB medium containing AMP and TET, and the cleared lysate prepared from each culture was analyzed by agarose gel electrophoresis. The substrate plasmids used were as follows: pGEN101, lanes 1 to 5; pGEN102, lane 6; pGEN103, lane 7; pGEN106, lane 8. The helper plasmids were as follows: pGEN21, lane 1; pGEN59, lane 2; pGEN77, lanes 3, 4, 6, 7, and 8; pGEN96, lane 5. The cleared lysates prepared from the LB cultures of the Km^r and Km^s derivatives of JM109(pGEN101)(pGEN77) were loaded in lanes 3 and 4, respectively. In lanes 1 and 2, pGEN21 and pGEN59 overlap pGEN101. oc, open circular DNA; chr, chromosome. (B) Cointegration of two plasmids, each carrying the Tn4651 res site. The donor strain DH5α (pGEN101R)(pGEN388)(pGEN77) was mated with HB101, and the Tc^r transconjugants were selected. Agarose gel electrophoresis of cleared lysates prepared from the donor strain (lane 1) and the transconjugant (lane 2) was done.

FIG. 6.

PCR detection of site-specific resolution. (A) Schematic drawing for PCR detection of the excised and nonreplicating DNA consisting of the res site and Km^r gene. White arrowheads indicate the primers. (B) Agarose gel electrophoresis of the PCR-amplified excised DNA fragments. PCR with primers 4Km1 and 4Km2 was performed by using the cleared lysate prepared from the JM109 derivative carrying the following combinations of plasmids: pGEN101 and pGEN21, lane 1; pGEN101 and pGEN59, lane 2; pGEN101 and pGEN77, lane 3; pGEN101 and pTrc99A, lane 4; pGEN101 and pGEN95, lane 5; pGEN101 and pGEN96, lane 6; pGEN102 and pGEN77, lane 7; pGEN103 and pGEN77, lane 8; pGEN106 and pGEN77, lane 9. In lane M, size markers are indicated in kilobases.