Polycistronic transcription of fused cassettes and identification of translation initiation signals in an unusual gene cassette array from Pseudomonas aeruginosa [version 3; referees: 2 approved]

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 ( ). Fused gene cassettes are generated attC by partial or total loss of the from the first cassette in an array, creating, in attC some cases, 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 gene cassette array, which gcu14-bla /aacA4 harbours a fused gene cassette represented by . The cassette bla /aacA4 array was analyzed by Northern blot and real-time reverse transcription-polymerase chain reaction (RT-PCR) in order to assess the transcription mechanism of fused cassette. Also, inverse bla /aacA4 polymerase chain reactions (PCR) were performed to detect the free circular forms of . The Northern blot and real time RT-PCR gcu14, bla and aacA4 revealed a polycistronic transcription, in which the fused cassette bla is transcribed as a unique gene, while (with a canonical /aacA4 gcu14 attC recombination site) has a monocistronic transcription. The cassette, gcu14 closer to the weak configuration of cassette promoter (PcW), had a higher transcription level than / , indicating that the cassette position bla aacA4 affects the transcript amounts. The presence of ORF-11 at , immediately attI1 preceding , and of a Shine-Dalgarno sequence upstream / gcu14 bla aacA4 composes a scenario for the occurrence of array translation. Inverse PCR generated amplicons corresponding to gcu14, gcu14-aacA4 and gcu14-bla / free circular forms, but not to and alone, aacA4 bla aacA4 indicating that the GES-1 truncated is not substrate of integrase activity attC 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. Referee Status: