Functional characterization of Tn1331 gene cassettes

Abstract

Objectives: The transposon Tn1331 possesses a region including three antibiotic resistance genes with the structure aac(6')-Ib-attC-aadA1-attI1*-bla(OXA-9)-attC, which potentially includes four gene cassettes. Experimental data on the mobility of fusion cassettes as well as those on mobility of cassettes in a genetic environment such as Tn1331, which lacks an integrase gene, are limited. Therefore, experiments using pJHCMW1, a plasmid harbouring this transposon, in the presence of IntI1 supplied in trans were carried out to define which cassettes are mobile in vivo.

Methods: In vivo excision of resistance genes was investigated in Escherichia coli cells harbouring pJHCMW1 and in a recombinant clone that included the intI1 gene under the control of the P(tac) promoter. Plasmid DNA was purified and subjected to PCR analysis, and DNA sequencing of PCR products was performed to determine whether excision had occurred.

Results and conclusions: In vivo recombination experiments showed that the fused aadA1-attI1*-bla(OXA-9)-attC gene cassette was excised in the presence of IntI1. The excision of a DNA fragment including aadA1-attI1* was also detected but at a lower frequency. The analysis of the latter recombination reaction showed that, although attI1* includes only a small fraction of the complete attI1 sequence, it is still used as a substrate by IntI1, albeit in a very inefficient manner.

Figure 1

(a) Genetic map of the Tn1331 region including genes aac(6′)-Ib, aadA1 and bla_OXA-9. The genes are shown in different patterns. Boxes with patterns represent attC loci and black boxes represent attI1* loci. The points of potential crossover reactions are indicated (downward arrows). Possible gene cassettes are indicated below the genetic map by bars of different patterns. The gene cassette including aadA1-attI1*-bla_OXA-9-attC is identified by a double box with two patterns. The primers are indicated by arrows of different patterns. The genetic map is at scale, but the primers and boxes are not. (b) Genetic map of the product of excision of the aadA1-attI1*-bla_OXA-9-attC gene cassette. (c) Genetic map of the product of excision of the aadA1-attI1* gene cassette.

Figure 2

(a and c) Agarose gel electrophoresis of amplicons obtained using plasmid DNA extracted from cells cultured in the conditions indicated in the text as template and the primers P1 and P2 (a) or P3 and P4 (c). The 1191 bp fragment (arrow) in the gel of (a) corresponds to the amplicon obtained when excision of the fragment aadA1-attI1*-bla_OXA-9-attC has occurred. The 568 bp fragment (arrow) in the gel in (c) corresponds to the amplicon obtained when excision of the fragment aadA1-attI1* has occurred. The line between the first and second lanes shows that they were not contiguous in the original gel. (b) Nucleotide sequence of the region surrounding the recombination site in the 1191 bp amplicon. (d) Nucleotide sequence of the region surrounding the recombination site in the 568 bp amplicon. The format of the fonts in the sequences shown in (b) and (d) corresponds to that in the genetic map shown in Figure 1(a).