Comparative study of class 1 integron and Vibrio cholerae superintegron integrase activities

Abstract

Superintegrons (SIs) and multiresistant integrons (MRIs) have two main structural differences: (i) the SI platform is sedentary, while the MRI platform is commonly associated with mobile DNA elements and (ii) the recombination sites (attC) of SI gene cassette clusters are highly homogeneous, while those of MRI cassette arrays are highly variable in length and sequence. In order to determine if the latter difference was correlated with a dissimilarity in the recombination activities, we conducted a comparative study of the integron integrases of the class 1 MRI (IntI1) and the Vibrio cholerae SI (VchIntIA). We developed two assays that allowed us to independently measure the frequencies of cassette deletion and integration at the cognate attI sites. We demonstrated that the range of attC sites efficiently recombined by VchIntIA is narrower than the range of attC sites efficiently recombined by IntI1. Introduction of mutations into the V. cholerae repeats (VCRs), the attC sites of the V. cholerae SI cassettes, allowed us to map positions that affected the VchIntIA and IntI1 activities to different extents. Using a cointegration assay, we established that in E. coli, attI1-x-VCR recombination catalyzed by IntI1 was 2,600-fold more efficient than attIVch-x-VCR recombination catalyzed by VchIntIA. We performed the same experiments in V. cholerae and established that the attIVch-x-VCR recombination catalyzed by VchIntIA was 2,000-fold greater than the recombination measured in E. coli. Taken together, our results indicate that in the V. cholerae SI, the substrate recognition and recombination reactions mediated by VchIntIA might differ from the class 1 MRI paradigm.

FIG. 1.

Alignment of VCR₁, VCR₂, attC_dfrA1, attC_aadA7, attC_orfA, attC_aadB, and attC_ereA site sequences. The 1L, 2L, 2R, and 1R sequences are enclosed in boxes. Plus signs and asterisks indicate conserved nucleotides found at all sites and at at least four of the sites examined, respectively. The vertical arrow indicates the recombination point in the 1R sequence.

FIG. 2.

Mutations introduced into the VCR₁-lacI^q-VCR₂ cassettes and recombination properties. 1R and 1L sequences are indicated by red type, and the nucleotides outside the 1R and 1L sequences are indicated by black type. Each individual mutation tested is on a single line and is identified by the designation of the cassette mutant, followed by the designation of the corresponding plasmid in parentheses; mutated bases are indicated by blue type and are underlined. The vertical arrows indicate the recombination points in the 1R sequences. Three asterisks indicate the VCR₁ and VCR₂ internal sequences of the original cassette. The deletion frequencies are expressed as percentages of the frequency established with the nonmutated VCR₁-lacI^q-VCR₂ synthetic cassette