Comparative Study of Class 1 Integron andVibrio choleraeSuperintegron 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-؋-VCR recombination catalyzed by IntI1 was 2,600-fold more efficient than attIVch-؋-VCR recombination catalyzed by VchIntIA. We performed the same experiments in V. cholerae and established that the attIVch-؋-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.

The Integron: Adaptation On Demand

“…On their side, it is also possible that MI-integrases show differential properties, such as distinct rates of activity or differences in the range of attC-sites recognized. Broader substrate recognition has indeed been observed to be the case for IntI1 compared with VchIntIA and IntIPstQ from Pseudomonas stutzeri (37,132). It is tempting to speculate that the success of class 1 integrons could be due, at least partly, to IntI1 recognizing a broader variety of attC sites and conferring access to a larger portion of cassette-encoded functions.…”

“…It has been observed that, contrary to the class I integron system, which recombines equally in E. coli and V. cholerae, the VchIntIA integrase of the V. cholerae CI recombines at a 2,600-fold higher rate the VCR sites in its original genomic background than in E. coli strains (37). Such results suggest either the involvement of one or more host factors in V. cholerae that would be absent or too divergent in E. coli strains, or the presence of an inhibitory factor in E. coli.…”

“…Nevertheless, in some cases cross-talk between two systems can occur. IntI1 from class 1 MIs can, for instance, integrate cassettes into attI2 and attI3 sites, albeit 100 times less efficiently than into attI1, but not at all into the attI site of the V. cholerae SI (37). Y-recombinase sites.…”

Natural transformation with synthetic gene cassettes: new tools for integron research and biotechnology

“…Previous studies have used excision assays (Collis & Hall, 1992;Drouin et al, 2002; Léon & Roy, 2003) or cointegrate assays (Biskri et al, 2005;Collis et al, 2002Collis et al, , 2001Holmes et al, 2003b;Martinez & de la Cruz, 1988) to quantify integron recombination. Improvements to these assays include the use of mobilizable suicide plasmids to deliver single-stranded recombination templates , and using quantitative PCR to simplify the cointegrate assay (Shearer & Summers, 2009;Wei et al, 2011;Yang et al, 2009).…”

“…Integrons are common in bacteria, and diverse in their phylogeny and predicted functions (Boucher et al, 2007a; Elsaied et al, 2007;Gillings et al, 2005;Holmes et al, 2003a;Nield et al, 2001;Rowe-Magnus & Mazel, 2001;Rowe-Magnus et al, 2003). Integrons increasingly seem to have a role as general-purpose agents of adaptation, not just enablers of antibiotic resistance (Koenig et al, 2009(Koenig et al, , 2011Rosewarne et al, 2010;Wright et al, 2008).Previous studies have used excision assays (Collis & Hall, 1992;Drouin et al, 2002; Léon & Roy, 2003) or cointegrate assays (Biskri et al, 2005;Collis et al, 2002Collis et al, , 2001Holmes et al, 2003b;Martinez & de la Cruz, 1988) to quantify integron recombination. Improvements to these assays include the use of mobilizable suicide plasmids to deliver single-stranded recombination templates , and using quantitative PCR to simplify the cointegrate assay (Shearer & Summers, 2009;Wei et al, 2011;Yang et al, 2009).…”