Bioinformatic and experimental characterization of SEN1998: a conserved gene carried by the Enterobacteriaceae-associated ROD21-like family of genomic islands

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.

Figure 1

The SEN1998 homologs encoded by the EARL GIs share high level of amino acid identity. Heatmap representation of the identity matrix resulting from the multiple sequence alignment of the amino acid sequences of SEN1998 and the homolog proteins encoded by the EARL family of GIs. The color represents identities in the range from 48.15% to 100.00%. Clusters of proteins with identities ≥ 90% and ≥ 70% are delimited by solid and dashed lines, respectively. The name of the bacterial strains harboring the EARL GIs are indicated at each side of the heatmap.

Figure 2

SEN1998 and its homologs encoded by plant and animal pathogenic bacteria cluster together according to phylogeny. Maximum likelihood unrooted tree of SEN1998 and its homologs encoded by the EARL GIs. Green and blue branches indicate proteins encoded by islands integrated in the chromosomes of plant and animal pathogenic bacteria, respectively. The node support calculated from 5,000 bootstraps is indicated for the basal nodes only.

Figure 3

The gene SEN1998 of ROD21 encodes a putative recombination directionality factor. (A) Multiple sequence alignment of the amino acid sequence of SEN1998 and characterized RDFs from phage (TorI, AlpA, Vis and Xis) and GI (Hef, VefA, VefB and VefC) origin. Conserved residues are colored according to the ClustalX coloring scheme. The black arrows indicate the conserved residues that has been recognized as important for the RDF function of TorI. (B) Percent identity of the aligned proteins regarding SEN1998, and isoelectric point. (C) Comparison of the secondary structures of SEN1998 (predicted), VefA (predicted), and TorI (experimentally determined). (D) Tertiary structures of the RDFs: Xis bound to DNA, (experimentally determined); TorI (experimentally determined) and SEN1998 (predicted).

Figure 4

The SEN1998 protein binds the two attachment regions of ROD21 with specificity. (A) Schematic representation of the target DNAs encompassing the left and right attachment regions of ROD21 used for the EMSAs. (B, C) EMSAs showing the binding of SEN1998 to both att regions, forming different complexes. The black and red arrows indicate the free and complexed target DNA. The multiple cloning site of plasmid pGEM-T Easy was used as a non-specific competitor DNA in C.

Figure 5

The excision of ROD21 increases during the stationary phase of growth as the expression of SEN1970 and SEN1998 decreases. ROD21 excision, and SEN1970 and SEN1998 expression patterns during the growth of Salmonella ser. Enteritidis P125109 in LB at 37 °C with shaking. The gray area represents the logarithmic phase of growth. Values are the mean ± SD of two independent experiments.

Figure 6

Effect of the absence and overexpression of SEN1998 and correlation of the excision with the SEN1970 and SEN1998 expression. (A) Excision levels of ROD21, represented as the percentage of the bacterial population with an empty attB site, in Salmonella ser. Enteritidis P125109, ΔSEN1998::frt and ΔSEN1998::frt pTrc-SEN1998, grown in the presence of different concentrations of IPTG. (B) mRNA levels of the integrase-encoding gene SEN1970 in the three strains, relative to the levels in P125109. Bars in A and B represent the mean of three biological replicates (individual points), at a specific IPTG concentration. (C) Scatter plot of the relative SEN1970 (left panel) and SEN1998 (right panel) mRNA levels versus the excision level in the same sample for the three Salmonella strains. The Pearson correlation coefficient and p-value are shown for each strain.