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The gene cassettes found in class 1 integrons are generally promoterless units composed by an open reading frame (ORF), a short 5’ untranslated region (UTR) and a 3’ recombination site ( attC ). Fused gene cassettes are generated by partial or total loss of the  attC  from the first cassette in an array, creating a fusion with the ORF from the next cassette. These structures are rare and little is known about their mechanisms of mobilization and expression. The aim of this study was to evaluate the dynamic of mobilization and transcription of the  gcu 14- bla  GES-1 / aac A4 gene cassette array, which harbours a fused gene cassette represented by  bla  GES-1 / aac A4. The cassette array was analyzed by Northern blot and real-time reverse transcription-polymerase chain reaction (RT-PCR) in order to assess the transcription mechanism of  bla  GES-1 / aac A4 fused cassette. Also, inverse polymerase chain reactions (PCR) were performed to detect the free circular forms of  gcu 14,  bla  GES-1  and  aac A4. The Northern blot and real time RT-PCR revealed a polycistronic transcription, in which the fused cassette  bla  GES-1 / aac A4 is transcribed as a unique gene, while  gcu 14 (with a canonical  attC  recombination site) has a monocistronic transcription. The  gcu 14 cassette, closer to the weak configuration of cassette promoter (Pc), had a higher transcription level than  bla  GES-1 / aac A4, indicating that the cassette position impacts the transcript amounts. The presence of ORF-11 at  attI1 , immediately preceding  gcu 14, and of a Shine-Dalgarno sequence upstream  bla  GES-1 / aac A4 composes a scenario for the occurrence of array translation. Inverse PCR generated amplicons corresponding to  gcu 14,  gcu 14- aac A4 and  gcu 14- bla  GES-1 / aac A4 free circular forms, but not to  bla  GES-1  and  aac A4 alone, indicating that the GES-1 truncated  attC  is not substrate of integrase activity and that these genes are mobilized together as a unique cassette. This study was original in showing the transcription of fused cassettes and in correlating cassette position with transcription.

f1000research

Polycistronic transcription of fused cassettes and identification of translation initiation signals in an unusual gene cassette array from Pseudomonas aeruginosa