Abstract

Horizontal gene transfer (HGT) plays a major role in adaptation and genome evolution in bacteria. The Enterobacteriaceae-associated ROD21-like (EARL) genomic islands (GIs), are a recently described family of horizontally acquired elements found in different species from the order Enterobacterales, including plant pathogens and clinically relevant bacteria, such as extraintestinal pathogenic Escherichia coli strains, carbapenem-resistant Klebsiella pneumoniae ST258 or Salmonella enterica serovars. Some EARL GIs encode putative virulence factors, however, most of their genes lack a predicted function. Among the genes carried by this family of islands there is a conserved excision/integration module which allows their excision as circular extrachromosomal elements, the first step for their horizontal transfer. Interestingly, it has been observed that the excision of ROD21, an EARL island carried by Salmonella ser. Enteritidis, Dublin, Gallinarum and Nitra, is involved in the pathogenesis of serovar Enteritidis, because mutant strains unable to excise ROD21 have a reduced capacity to colonize the internal organs of infected mice. As excision is important for the spread of the EARL GIs, the pathogenesis of Salmonella ser. Enteritidis, and possibly for other bacterial pathogens, we aimed to gain insight about the genes involved in this process. Therefore, we studied a gene conserved across the EARL GIs, SEN1998, whose overexpression in Salmonella ser. Enteritidis was previously found to increase the excision of ROD21. Analysis of the predicted primary, secondary and tertiary structures of the SEN1998 protein showed that it is a small basic protein of 72 amino acids that share the main features found in recombination directionality factors (RDFs) from phage and island origin, including a predicted winged-helix DNA-binding domain. Then, we cloned and expressed SEN1998 to purify its product. The recombinant SEN1998 (rSEN1998) protein was used in binding assays using the left and right attachment (att) regions of ROD21, which are involved in the assembly of the macromolecular DNA-protein complex that carries out excision. Interestingly, we found that rSEN1998 specifically binds both att regions with different affinities, forming different complexes that suggest the presence of more than one binding site. Our results strongly suggest that SEN1998 and their homologs in the EARL GIs encode RDFs, and may contribute to the study of HGT. Further experiments assessing the effect of the deletion of SEN1998 in the excision of ROD21 are in progress.