Abstract
Genomic islands (GIs) are horizontally transferred elements that shape bacterial genomes and contributes to the adaptation to different environments. Some GIs encode an integrase and a recombination directionality factor (RDF), which are the molecular GI-encoded machinery that promotes the island excision from the chromosome, the first step for the spread of GIs by horizontal transfer. Although less studied, this process can also play a role in the virulence of bacterial pathogens. While the excision of GIs is thought to be similar to that observed in bacteriophages, this mechanism has been only studied in a few families of islands. Here, we aimed to gain a better understanding of the factors involved in the excision of ROD21 a pathogenicity island of the food-borne pathogen Salmonella enterica serovar Enteritidis and the most studied member of the recently described Enterobacteriaceae-associated ROD21-like family of GIs. Using bioinformatic and experimental approaches, we characterized the conserved gene SEN1998, showing that it encodes a protein with the features of an RDF that binds to the regulatory regions involved in the excision of ROD21. While deletion or overexpression of SEN1998 did not alter the expression of the integrase-encoding gene SEN1970, a slight but significant trend was observed in the excision of the island. Surprisingly, we found that the expression of both genes, SEN1998 and SEN1970, were negatively correlated to the excision of ROD21 which showed a growth phase-dependent pattern. Our findings contribute to the growing body of knowledge regarding the excision of GIs, providing insights about ROD21 and the recently described EARL family of genomic islands.
Highlights
Syringae pathovar phaseolicola and Salmonella enterica serovar Enteritidis, in which changes in the expression of genes inside the islands have been observed in response to the integrated/excised state of the G I10,11
The two attachment regions that flank the integrated Lambda phage, attL and attR, are the result of the recombination between the attB insertion site in the bacterial chromosome and the attP region in the circular phage DNA; attL and attR are populated by several binding sites for the different proteins involved in the assembly of the excisive intasome, the high-order nucleoprotein complex required for the excision of the prophage[12]
While the Lambda integrase participates in the integration and excision reactions, the phageencoded recombination directionality factor (RDF) Xis is key for excision, since it promotes the assembly of the excisive intasome, inhibiting integration[15,16]
Summary
Syringae pathovar phaseolicola and Salmonella enterica serovar Enteritidis, in which changes in the expression of genes inside the islands have been observed in response to the integrated/excised state of the G I10,11. Lambda- and host-encoded proteins bind the attL and attR regions to bend the DNA and promote the intasome assembly, allowing the phage-encoded tyrosine recombinase, the integrase, to catalyze a site-specific recombination between the direct repeated sequences (DRSs) located at the end of each attachment region[13,14]. Enteritidis, since mutant strains unable to excise ROD21 had a significantly reduced capacity to colonize the spleen, liver and gallbladder compared to the wild-type strain in which ROD21 is excised normally[11,25] This phenomenon was hypothesized to be due to a regulatory mechanism in which gene expression within ROD21 is modulated by the supercoiling of the island, which may differ between its integrated and excised states, as observed for the PPHGI-1 island of Pseudomonas syringae pv. We describe the main features of SEN1998 and its protein product through bioinformatic and experimental analyses
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