IntI2 integron integrase in Tn7

Abstract

Integrons can insert and excise antibiotic resistance genes on plasmids in bacteria by site-specific recombination. Class 1 integrons code for an integrase, IntI1 (337 amino acids in length), and are generally borne on elements derived from Tn5090, such as that found in the central part of Tn21. A second class of integron is found on transposon Tn7 and its relatives. We have completed the sequence of the Tn7 integrase gene, intI2, which contains an internal stop codon. This codon was found to be conserved among intI2 genes on three other Tn7-like transposons harboring different cassettes. The predicted peptide sequence (IntI2*) is 325 amino acids long and is 46% identical to IntI1. In order to detect recombination activity, the internal stop codon at position 179 in the parental allele was changed to a triplet coding for glutamic acid. The sequences flanking the cassette arrays in the class 1 and 2 integrons are not closely related, but a common pool of mobile cassettes is used by the different integron classes; two of the three antibiotic resistance cassettes on Tn7 and its close relatives are also found in various class 1 integrons. We also observed a fourth excisable cassette downstream of those described previously in Tn7. The fourth cassette encodes a 165-amino-acid protein of unknown function with 6.5 contiguous repeats of a sequence coding for 7 amino acids. IntI2*179E promoted site-specific excision of each of the cassettes in Tn7 at different frequencies. The integrases from Tn21 and Tn7 showed limited cross-specificity in that IntI1 could excise all cassettes from both Tn21 and Tn7. However, we did not observe a corresponding excision of the aadA1 cassette from Tn21 by IntI2*179E.

FIG. 1.

Maps of the integron regions in Tn7 and in the related transposons Tn1825 and Tn1826. Integron sequences are represented by boxes. Black boxes indicate the conserved sequence (5′-CS-2) with the integrase gene, intI2. Cassettes are shown by white boxes. Leftward-pointing filled arrowheads denote inverse core sequences inside the cassettes, and rightward-pointing open arrowheads denote core sequences where DNA strand transfer occurs. Vertical lines at cores indicate cassette borders. The PinAI and EcoRI restriction enzyme cut sites used for cloning the cassette regions are indicated. Nucleotide sequences determined in this study are underlined. Newly determined sequences are indicated with heavy black lines, while heavy gray lines indicate previously determined sequences that were checked in the present study. Vertical boxes denote the 22-bp repeats, L1-3, for transposase binding, which are clustered close to the left Tn7 end (2).

FIG. 2.

(A) The nucleotide sequence of the left end of transposon Tn7 containing intI2 and the new ORFX (translated) cassette. L1 to L3 indicate the multiple repeats clustered at the end of the transposon (2). Stop codons in the intI2 gene are in boldface and are marked with three dots below the sequence. The start codon of the gene is shown with an arrow. The G in the GTT sequences at the ORFX cassette borders are underlined by double dashed lines. Underlined sequences mark the core and inverse core in the ORFX cassette. The peculiar repetitions in ORFX are marked with horizontal lines above the sequence and are indexed by r1 to r7. The sequence at positions 4782 to 4856 is identical to a sequence in Arabidopsis (M55553). (B) Comparison of the amino acid sequences of the translated intI1, intI2, intI3, and intI4 (also called VchintIA). The well-conserved domains 1 and 2, with the four amino acids marked that are highly conserved among all members of the tyrosine integrase family, are underlined. (C) Nucleotide sequences and translated amino acid sequences of the 6.5 repetitions in ORFX. The sequence from positions 940 to 1970 was found to be identical in Tn1825 and Tn1826.

FIG. 3.

Site-specific recombination between cassette ends promoted by IntI1 or IntI2*179E from cloned integron fragments of class 1 and class 2 integrons. Recombination was detected by divergent and convergent PCR as described in Materials and Methods. The ability of the different integrases to promote excision of the tested cassettes is denoted by plus and minus signs. Identical results were obtained with divergent and convergent PCR assays. (A) Plasmid 670 carrying the cassette region from Tn7 (class 2 attI site). (B) Plasmid 678 with the cassette region from Tn7 deleted for the dfrA1 and sat cassettes and plasmid 2280 carrying the cassette region from Tn21 (Class 1 attI site). White boxes indicate cassettes, and arrows indicate the positions of the PCR primers used in this study. Black boxes indicate the conserved sequence (5′-CS-2) from the class 2 integron of Tn7, while the conserved sequence from the class 1 integron in Tn21 is hatched (5′-CS-1). The primers used are denoted as follows: p1, Tn7SRS-107; p2, cup11; p3, cdo11; p4, sat1do2; p5, sat1up2; p6, cup2; p7, cdo2; p8, Tn7CRS+185; p9, Tn7Fling; p10, reverse; p11, pSph; and p12, pHin.

FIG. 4.

Structures of cloned integron fragments and recombination rates detected with the mating-out assay as described in Materials and Methods. Integron fragments from either Tn21 or Tn7 were tested for recombination with wild-type R388 and R388Δ1 in the presence of IntI1 or IntI2*179E. Conserved sequences from class 1 integrons are hatched (5′-CS-1) and from class 2 are in black boxes (5′-CS-2). Open white boxes indicate cassettes. The lengths of 5′ and 3′ flanks from the closest cassette border are given above the cloned integron fragments. Recombination frequencies are given as mean transfer frequency of million transferred R388 or R388Δ1, with ranges given within brackets. All experiments were repeated at least four times. Only background recombination rates were detected between the tested pSU18 clones and R388Δ1 with IntI2*179E.