Class 1 integrons are capable of inserting, excising and rearranging gene cassettes by a site-specific recombination mechanism. These assembly platforms can also act as expression systems due to the presence of a promoter region (Pc), which drives the expression of genes captured by integron ¹ . Moreover, naturally occurring integrons may have a second promoter (P2), which is activated by the insertion of three G residues between -35 and -10 hexamers ¹ . Gene cassettes are generally promoterless units associated with a recombination site ( attC or 59-be), which confers the ability of each structure to be mobilized independently ² . In addition, attC sites regulate the translation of downstream cassettes due to their peculiar sequences composed by imperfect inverted repeats. The formation of stem-loop structures by attC sites prevents ribosome progression throughout mRNA, reflecting in a decreased expression of more distal genes regarding Pc ³ . Although rare, fused cassettes may be generated by partial or total loss of the first attC, retaining both complete coding regions and, therefore, creating permanent gene arrays comparable to bacterial operons ⁴ . The functionality of such structures has been indirectly inferred by the resistance profile of transformants carrying the fusion ⁵ ; however, the transcription itself has never been verified.

This study showed the dynamics of fused cassette mobilization, the co-transcription of the gcu14- bla _GES-1/ aacA4 cassette array and the effect of cassette position on transcription levels in Pseudomonas aeruginosa wild lineages carrying class 1 integrons. Moreover, the presence of translation signals in this gene cassette array was determined.

An unknown Open Reading Frame (ORF), gcu14, followed by the fused cassette bla _GES-1/ aacA4, created by partial loss of GES-1 attC were present in integrons from clinical P. aeruginosa isolates (PS1 and PS26) ⁶ . Total RNA was extracted and purified according to the manufacturers instructions with the SV 96 Total RNA Isolation System (Promega). Northern blot using 7 μg of total RNA from PS1 and PS26 was performed in order to detect the transcript originated from gcu14- bla _GES-1/ aacA4 cassette array. After electrophoresis in a denaturing-formaldehyde 1.5% agarose gel, the total RNA was transferred to the Hybond-N ⁺ nylon membrane (GE Healthcare) by upward capillary transfer. An amplicon of 519bp corresponding to part of the bla _GES-1 gene was used as a probe (Table 1) in hybridization assay. The GES probe was labeled with the AlkPhos Direct Labelling kit (GE Healthcare) and hybridized with the target RNA immobilized on the Hybond-N ⁺ membrane as recommended. The chemiluminescence was detected with the CDP-Star detection reagent (GE Healthcare) according to manufactures. Immediately after applying the detection reagents, the blot was drained, incubated five minutes at room temperature and exposed to the Hyperfilm ECL (GE Healthcare) for 60 minutes at room temperature.

In order to verify whether the relative position of gene cassettes on the variable region plays a role in transcription level, real-time RT-PCR reactions using the TaqMan System (Applied Biosystems) were performed with primers and probes detailed in Table 1. The single-copy ribosomal rpsL gene of the P. aeruginosa chromosome was amplified by PCR (Table 1) and used as a reference gene for normalization. The relative quantification (RQ) results were presented as ratios of gene transcription between the target gene (cassettes) and the reference gene ( rpsL), which were obtained according to the following equation: RQ=2 ^-ΔCT, where CT is the value corresponding to the crossing point of the amplification curve with the threshold and ΔCT=CT target gene minus CT reference gene. The effect of cassette position on gene transcription was considered significant when the ratios obtained between RQ values (RQ value of cassette 1/RQ value of cassette 2) were ≥2.0, taking into account the standard deviation intervals.

In order to induce cassette excision from integrons, PS1 and PS26 strains ⁶ were submitted to thermal stress during the log growth phase to induce integrase activity. Cells were grown on Luria-Bertani (LB) broth medium (OXOID) at 37ºC for two hours. subsequently, the bacterial cultures were submitted to a heat shock at 4ºC for 30 minutes and immediately incubated at 42ºC for another 30 minutes. Briefly, the total DNA from PS1 and PS26 cultured under thermal stress were obtained with the Wizard Genomic DNA purification kit (Promega) following manufacturer recommendations and used as templates in inverse PCR reactions. The inverse PCR was performed with primers facing outwards towards the ends of gcu14, bla _GES-1 and aacA4 so that only circular gene cassette configurations would be amplified. The reactions target the circular forms of gcu14, bla _GES-1, aacA4 and bla _GES-1/ aacA4 fusion by using the primers and combinations described in Table 1. The inverse PCR was performed using Platinum Taq DNA Polymerase reagents (Invitrogen), and the following components were added to a sterile 0.2-mL tube: 5 µL of 10X PCR buffer (1X final concentration); 1 µL of 10mM dNTP mixture (0.2 mM each); 1.5 µL of 50mM MgCl ₂ (1.5 mM final concentration); 2 µL of 15 µM of each primer (30 µM each); 100 ng of template DNA; 0.3 µL of Platinum Taq DNA Polymerase (1U final concentration). The tubes were incubated in the Eppendorf MasterCycler (Eppendorf) at 94°C for 2 minutes and PCR amplification was performed in 40 cycles consisting of: 94°C for 30 seconds; 55 °C for 30 seconds; and 72°C for 3 minutes. The amplicons generated with the inverse PCR were purified using Wizard SV Gel and PCR Clean-Up system kit (Promega) and directly sequenced on both strands. Sequencing reactions were performed with Big Dye Terminator RR Mix (Applied Biosystems) in an ABI 3730 XL DNA Analyzer (Applied Biosystems). Nucleotide sequences were compared to those available in the GenBank database accessible on the National Center for Biotechnology Information website ( http://www.ncbi.nlm.nih.gov). All primers used in PCR, sequencing and real time RT-PCR are described in Table 1.

Analyses in silico were performed to search for a potential promoter for gcu14 gene cassette. The 5’UTR from gcu14 were submitted to the promoter predictor programs Neural Network for Promoter Prediction version 2.2 (Berkeley Drosophila Genome Project, http://www.fruitfly.org/index.html) and BPROM (SoftBerry, http://linux1.softberry.com/berry.phtml). Results with the highest scores were selected as candidates for a putative promoter.

The integrons analyzed in this study harbored a rare weak Pc configuration (PcW _TGN-10), which presents a C to G mutation 2 bp upstream of the −10 hexamer that causes an abrupt decrease in promoter strength as reported previously ⁷ . Considering that transcription initiates from the Pc promoter placed upstream the gcu14- bla _GES-1/ aacA4 cassette array, both monocistronic and full length polycistronic transcripts could be identified. In fact, Northern blot and hybridization assays revealed a unique signal of approximately 2,300 bases which corresponds to the co-transcription of the entire array ( gcu14- bla _GES-1/ aacA4 ) (Figure 1). This result is in agreement with previous work in which the occurrence of transcripts containing more than one gene cassette was observed by Northern blot analysis ¹ . Moreover, this finding gives support to the lack of attC function in terminating transcription of downstream gene cassettes as demonstrated previously ³ .

This fusion retained both entire coding regions and suffered partial loss of 91 bp at the GES-1 attC site ( DQ236170) ⁶ . Previous studies demonstrated that the attC region flanked by Left Hand (LH) and Right Hand (RH) domains, which is an imperfect inverted repeat and is missing in the bla _GES-1 recombination site, is crucial for cassette mobilization ⁸ . Taking into account that the region responsible for stem-loop formation was missing in GES-1 attC and the participation of this site in terminating translation ³ , our findings indirectly suggested that bla _GES-1 and aacA4 translation is occurring in a unique step.

Gene cassettes can be found inserted in integrons or in other secondary sites or free in the cytoplasm as a closed circle, in which the 5’ end (5’ UTR) and the attC recombination site are covalently linked ⁹ . As demonstrated previously, several stress conditions could evoke the activation of the SOS response resulting in integron-integrase expression ¹⁰ . Therefore, under stress, the integrase activity increases, favoring the occurrence of integration/excision/rearrangements events. Since the excision event depends on the recognition of the LH and RH domains of the attC site, and that these regions are missing in GES-1 attC, it is expected that the bla _GES-1/ aacA4 excision occurs only at the aacA4 attC site, and that this structure is excised together as a unique cassette.

Positive results were obtained for the gcu14, gcu14- aacA4 and gcu14- bla _GES-1/ aacA4 circular forms, but not for bla _GES-1 and aacA4 alone, showing that the GES-1 attC is not functional and that the fused gene cassette is excised as a unique cassette. Sequencing assessed the recombination point where excision occurred, confirming the occurrence of free circular forms. The lack of activity of a truncated attC had also been observed before when associated with aadA10 ¹¹ . However, Ramirez and colleagues ¹² showed that the integrase was able to recognize and mediate excision of a truncated site associated to aadA1, indicating that the genetic context of such truncated sites could influence their role in IntI1 recognition and mobilization.

The relative quantification performed by real time RT-PCR revealed that PS1 and PS26 presented very similar RQ values for gcu14- bla _GES-1/ aacA4 transcription (Figure 2). This result was expected since integrons from these two strains have the same backbone, including the Pc promoter, and are at the same genetic environment ⁶ .

gcu14, the first cassette in integrons with the weak PcW _TGN-10 configuration, presented approximately two-fold higher transcription when compared to bla _GES-1 and aacA4 separately or when the fused cassette bla _GES-1/ aacA4 was considered (Figure 2). The same RQ value obtained for bla _GES-1, aacA4 and the fusion reveals that these two ORFs are transcribed as a unique gene. The lower transcript amount of bla _GES-1/ aacA4 compared to gcu14 lies on the distance between these gene cassettes and Pc, which is one of the determinants influencing cassette transcription ^{1, 10} , and it shows the effect of cassette position on expression levels.

A putative promoter for gcu14 (-35 TTGATG [17 bp] -10 TGTTAC) was found 45 bp upstream from its start codon. Therefore, the increased gcu14 transcription could have resulted from a synergistic effect of this putative promoter with Pc. Considering that this putative promoter was found at attI1, which is highly conserved among class 1 integrons, it can be suggested that such a promoter is able to drive transcription of any cassette placed in the first integron position. Moreover, the ORF-11, which enhances the translation efficiency of downstream TIR (translation initiation region)-deficient cassettes inserted in integrons ¹³ , was found at the attI1 region preceding the TIR-deficient gcu14 gene cassette. This ORF contained its own Shine-Dalgarno (SD) sequence placed 8 bp upstream of the ATG codon. The ribosome at the ORF-11 stop codon could therefore be carried along the mRNA by lateral diffusion, reinitiating translation at the gcu14 start codon. A potential SD sequence was identified 10 bp upstream of the fused cassette bla _GES-1/ aacA4. In addition, the loss of the GES-1 attC region, which is involved in stem-loop formation, may enhance the chances of aacA4 translation, since this attC no longer constitutes a physical barrier to ribosome progression ³ . This deletion also then brings the second gene ( aacA4) closer to Pc ¹⁴ . Together, these findings create a scenario for the occurrence of gcu14- bla _GES-1/ aacA4 expression in PS1 and PS26, which then provides a possible explanation for their resistance profile to β-lactams and aminoglycosides that has been observed elsewhere ⁶ .

Fused cassettes have been found in class 1 integrons ^{5, 11, 12, 15– 18} ; however, the transcription of such structures has never been addressed. This work showed the transcription pattern of a fused cassette as a polycistronic mRNA and that these unusual structures are excised as a unique cassette.