## 2 Transcriptional Regulation in Integrons
### The Role of Cassette-Borne Promoter on Gene Cassette Transcription
The in vitro assays to assess their relative transcription revealed that both gene cassettes produced similar transcript amounts, indicating the presence of an internal promoter in _qnrVC1_. This gene cassette presented an unusually long 5' UTR of 216 bp, and in silico approaches revealed the presence of a potential promoter in this region placed 29 bp upstream _qnrVC1_ start codon. This promoter (P\({}_{qnrVC}\)) was related to _E. coli_\(\sigma^{70}\)-dependent promoters, preserving 8 of the 12 canonical nucleotides of the -35 and -10 hexamers. The determination of _qnrVC1_ TSS by 5'RACE revealed that P\({}_{qnrVC}\) was 14 bp upstream the +1 position of the transcript, confirming that this promoter was responsible for driving _qnrVC1_ transcription. Moreover, it was demonstrated that P\({}_{qnrVC}\) was able to promote the expression of the _cat_ gene in promoterless-probe vectors, conferring chloramphenicol resistance in vitro. These findings, together with the production in vivo of fluorescence by _E. coli_ cells due to the transcriptional fusion of P\({}_{qnrVC}\) with the _gff_ reporter gene, proved that P\({}_{qnrVC}\) was functionally active and was responsible for _qnrVC1_ expression and quinolone resistance manifestation [69]. It was also demonstrated that P\({}_{qnrVC}\) versions were distributed among other _qnrVC_ alleles [10; 11; 69; 70; 71], ensuring their expression and emergence of resistance phenotypes in distinct genetic contexts and species. In the context of CIs, cassettes placed in very distal positions relative to Pc, which would be silenced, might be efficiently expressed by the presence of internal promoters. In fact, functional studies of large CIs from different species have demonstrated that a great proportion of gene cassettes, mainly those coding for toxin-antitoxin (TA) systems, were efficiently transcribed due to the presence of cassette-borne promoters, such as _brmT-brmA_, _vapC-abrB/mazE/spoVT_, and _relE-parE_[4; 72; 73; 74; 75]. TA systems avoid the entrance of mobile genetic elements by horizontal gene transfer and contribute to the genome stabilization [72; 73; 74]. For example, the TA systems encoded by _phd-doc_ and the _ccd\({}_{V\!fi}\)_ cassettes found in _V. cholerae_ and _V. fischeri_ CIs were efficiently expressed by their own active promoters. Similarly, another study demonstrated that the TA loci _relBE1_ and _parDE1_ cassettes carried in the _V. vulnificus_ CI were expressed in vivo and these active TA systems counterbalanced the extent of deletions catalyzed by the integrase [73]. The constitutive expression of such TA cassettes observed in different _Vibrio_ species indicated that these systems have a relevant role in the stabilization of CI-associated gene cassettes by minimizing large-scale cassette loss without hindering the microevolution of these genetic platforms mediated by integrase activity [73; 74]. Moreover, additionally to TA gene cassettes, other studies experimentally demonstrated that most of the gene cassettes in large CI arrays were transcribed by their own promoters. For example, it was verified that most of the 116-gene cassettes in the CI array of _V. rotiferianus_ DAT722 were widely transcribed due to the presence of numerous and diverse internal promoters across the array and that these cassettes were differentially transcribed in response to promoter strengths and to variations in environmental conditions such as thermal and oxidative stress. Therefore, these bacteria harboring CIs have the potential to benefit from the concomitant manifestation of multiple functions irrespective of cassette location within the array. Consequently, the presence of larger functional cassette arrays can provide an increased repertoire of adaptive capabilities, conferring complex phenotypes and multiple selective advantages to the host organism, which could explain the prevalence of CIs in the environment [76].