Inverse correlation between promoter strength and excision activity in class 1 integrons

Abstract

Class 1 integrons are widespread genetic elements that allow bacteria to capture and express gene cassettes that are usually promoterless. These integrons play a major role in the dissemination of antibiotic resistance among Gram-negative bacteria. They typically consist of a gene (intI) encoding an integrase (that catalyzes the gene cassette movement by site-specific recombination), a recombination site (attI1), and a promoter (Pc) responsible for the expression of inserted gene cassettes. The Pc promoter can occasionally be combined with a second promoter designated P2, and several Pc variants with different strengths have been described, although their relative distribution is not known. The Pc promoter in class 1 integrons is located within the intI1 coding sequence. The Pc polymorphism affects the amino acid sequence of IntI1 and the effect of this feature on the integrase recombination activity has not previously been investigated. We therefore conducted an extensive in silico study of class 1 integron sequences in order to assess the distribution of Pc variants. We also measured these promoters' strength by means of transcriptional reporter gene fusion experiments and estimated the excision and integration activities of the different IntI1 variants. We found that there are currently 13 Pc variants, leading to 10 IntI1 variants, that have a highly uneven distribution. There are five main Pc-P2 combinations, corresponding to five promoter strengths, and three main integrases displaying similar integration activity but very different excision efficiency. Promoter strength correlates with integrase excision activity: the weaker the promoter, the stronger the integrase. The tight relationship between the aptitude of class 1 integrons to recombine cassettes and express gene cassettes may be a key to understanding the short-term evolution of integrons. Dissemination of integron-driven drug resistance is therefore more complex than previously thought.

Figure 1. General structure of a class 1 integron.

Coding sequences are indicated by arrows, attC cassette recombination sites by triangles, the integron recombination site attI1 by a circle, and the gene cassette promoters Pc and P2 by broken arrows. Dotted vertical bars represent gene cassette boundaries.

Figure 2. Strength of class 1 Pc variants.

(A) Pc promoter strength was estimated by measuring the β-galactosidase activity of Pc-lacZ transcriptional fusions. (B) β-galactosidase activities of Pc-lacZ fusions for different Pc variants in either their wild-type configuration (white bars) or bearing the TGN-10 motif (grey bars). (C) Same as (B), but for Pc variants in combination with P2. PcS* designates an artificially mutated PcS variant that was combined with P2 to serve as a control for specific P2 promoter activity. At least five independent assays were performed for each variant and in each experiment. Error bars indicate the standard error of the mean.

Figure 3. Recombination activities of the main IntI1 variants.

IntI1 excision recombination activity was estimated by determining the frequency of emergence of the tobramycin resistance phenotype as a result of recombination between the attC sites of the attC_aadA7-catT4-attC_VCR-aac(6′)-Ib array, leading to the deletion of the synthetic cassette catT4-attC_VCR and expression of the tobramycin resistance gene aac(6′)-Ib. IntI1 integration recombination activity was estimated by determining the frequency of emergence of cointegrate of two plasmids, one carrying an attI site and the other an attC site. Error bars indicate the standard error of the mean for at least seven independent assays.