## 2 Transcriptional Regulation in Integrons
### Interplay between Integrase and Gene Cassette Expression in Class 1 Integrons  
#### 2.6.1 Integrase Activity Is Linked and Inversely Correlated to Gene Cassette Expression  
As extensively discussed above, dozens of Pc variants may naturally occur in class 1 integrins due to point mutations in the -35 and -10 hexamers [26]. Since the Pc promoter is found embedded in the 5\({}^{\prime}\) end of the _intII_ coding sequence, the polymorphisms that give rise to the different Pc promoter variants may also lead to non-synonymous mutations in the _intII_ gene, resulting in amino acid changes in integrase. These alterations in the class 1 integrase interfere with its recombination activity and determine different catalytic _intII_ variants with varying efficiencies of excision mediated by _attC X attC_ recombination. The same IntII variant may be defined by different Pc promoters, in which 13 Pc versions determine 10 variants of IntII. Three of these 10 integrase versions, with amino acid changes occurring at positions 32 and 39 (IntII1R32_H39, IntIIR32_N39, and IntII1P32_H39) represent the great proportion of IntI variants. [PERSON] et al. [26] estimated the ability of these integrase variants in excising gene cassettes. They revealed that the substitution of an arginine (R) by a proline (P) at residue 32 and of histidine (H) by an asparagine (N) at residue 39 led to a remarkable reduction in integrase excision efficiency. Comparisons with the IntIA from _V. cholerae_ CI revealed that these amino acid positions were within an \(\alpha\)-helix involved in _attC_ binding [114], which is also conserved in the predicted structure of IntII [115]. Therefore, amino acid changes at positions 32 and 39 probably disturb the _attC_ binding by IntII, resulting in the low excision efficiency observed for these integrase variants. On the other hand, such amino acid changes had a less relevant impact on the efficiency of gene cassette integration mediated by IntII variants, indicating that _attI_ X _attC_ (integration) and _attC_ X _attC_ (excision) recombination reactions may involve different regions of the integrase [26]. Therefore, an inverse correlation between Pc promoter strength and the integrase excision activity was observed, since the polymorphisms that defined weaker Pc promoters also defined more active integrase enzymes, while those determining the strongest Pc configuration led to integrase variants with lower activity. The IntIIR32_H39, the variant with the most efficient excision activity, correlated with the presence of Pc configurations (PcW and PcH1) involved with weak transcriptional strength. In this case, the low expression level of resistance genes conferred by weak Pc versions might be compensated by the high excision rates evoked by the variant IntIIR32_H39, which confers a remarkable ability of cassette reassortment that may bring previously silenced cassettes closer to Pc, enabling bacteria to adapt more rapidly to harsh environments. On the other hand, in a challenger scenario of heavy antibiotic pressure, stronger Pc configurations, such as PcS and PcW\({}_{\text{TGN-10}}\), are selected in order to express antibiotic resistance gene cassettes more efficiently, in detriment of IntII excision activity, leading to the maintenance of advantageous cassette arrays. This assumption is in accordance with the fact that integrins carrying the integrase variants IntIIR32_N39 or IntII1P32_H39 generally have arrays with a greater number of gene cassettes than those harboring IntI1R32_H39, since the formers are less efficient in excising gene cassettes [26]. A previous study evaluated the efficiency of rearrangements mediated by different IntI1 variants under antibiotic selective pressure. It was demonstrated that, independently of the integrase variant present, the IntI1 was able to perform multiple cassette excision events to bring a transiently silenced _catB9_ gene cassette closer to Pc in response to chloramphenicol presence.