Integron attI1 Sites, Not Riboswitches, Associate with Antibiotic Resistance Genes

In a recent publication in Cell (Jia et al., 2013), the authors conclude from bioinformatics, genetic, and biochemical analyses that a conserved sequence element associated with antibiotic resistance genes represents a novel widespread riboswitch class for certain aminoglycosides. Below, we point out previously reported functions for this element that were not discussed by the authors, and we note other issues with the validation experiments that raise significant concerns about the conclusions of the study. Most riboswitches contain highly conserved ligand-binding domains, and therefore the identification of consensus sequence and structure models is critical in providing support for proposed riboswitch classes. Jia et al. describe a conserved sequence element that is frequently associated with aminoglycoside acetyl transferase (aac) and aminoglycoside adenyl transferase (aad) genes, and they ultimately conclude that it corresponds to mRNA leader sequences with riboswitch functions. However, the consensus sequence that they describe was already known to be a key component of integrons, serving as a recombination site (attI) that facilitates the exchange of gene cassettes sometimes involved in antibiotic resistance (Mazel, 2006). Sequences in their alignment (Data S1) correspond specifically to the attI1 class of recombination sites (Partridge et al., 2000), and the locations of these sites— within class 1 integrons, between the intI1 gene encoding the integrase and the integron cassettes—are consistent with a function in recombination. Importantly, the most highly conserved segments in the alignment, which contain the sequence defined by the authors as the riboswitch aptamer domain, precisely encompass the regions of the attI1 DNA that interact with the integrase and that are necessary for recombination to occur (Recchia et al., 1994; Collis et al., 1998; Gravel et al., 1998). Moreover, there are no segments other than those corresponding to the attI1 site that are universally conserved among the aligned sequences. Thus, if these sequences also function at the level of RNA as aminoglycoside riboswitches (in addition to their known functions at the DNA level as specialized recombination sites), the conserved domains responsible for each independent function would have to be perfectly coincident. Although Jia et al. mention in the Discussion that their reported sequences are ‘‘constituent(s) of the integron cassette system,’’ they never state that these sites have biochemical functions that were already well understood. Given the previously established function of this element, it seems unlikely that the conserved nucleotides are also important for riboswitch function. This concern is reinforced by the fact that the locations of attI1 sequences are not limited to regions near aminoglycoside resistance genes, as implied by the data depicted in Figure 1C. Integrons containing attI1 sites commonly contain gene cassettes involved in resistance to a wide range of antibiotics, including trimethoprim, rifampicin, chloramphenicol, tetracycline, erythromycin, sulfonamides, quinolones, quaternary ammonium compounds, and various b-lactams (Moura et al., 2009). It seems counterintuitive for the optimal expression of these myriad antibiotic resistance genes to be contingent on the function of a riboswitch that responds only to a limited number of aminoglycoside derivatives. In addition, there are numerous instances of aminoglycoside resistance genes occurring in contexts apart from integrons, many examples of which are listed by the authors in Table S1A. In such instances, the aminoglycoside resistance genes are never observed to be associated with attI1 sites. It is only when these genes are contained within