SEN1990 encodes a transcriptional regulator from Salmonella enterica serovar Enteritidis that modulates the expression of the SPI-17 encoded gene oafB

Excisable Genomic Islands (EGIs) are horizontally acquired genetic elements that harbor an array of genes with diverse functions. ROD21 is an EGI found integrated in the chromosome of Salmonella enterica serovar Enteritidis (Salmonella ser. Enteritidis). While this island is known to be involved in the capacity of Salmonella ser. Enteritidis to cross the epithelial barrier and colonize sterile organs, the role for most of ROD21 genes remains unknown, and thus identification of their function is fundamental to understand the impact of this EGI on the bacterium pathogenicity. Therefore, in this work we used a bioinformatical approach to evaluate the function of ROD21-encoded genes and delve into the characterization of SEN1990, a gene encoding a putative DNA-binding protein. We characterized the predicted structure of SEN1990, finding that this protein contains a three-stranded winged helix-turn-helix (wHTH) DNA-binding domain. Additionally, we identified homologs of SEN1990 among other members of the EARL EGIs. Furthermore, we deleted SEN1990 in Salmonella ser. Enteritidis, finding no differences in the replication/maintenance of the excised ROD21, disconfirming the previous Refseq annotation of the protein. High-throughput RNA-sequencing was carried out to evaluate the effect of the SEN1990 absence on the bacterium global transcription. We found a downregulated expression of oafB, an SPI-17-encoded acetyltransferase involved in O-antigen modification, which was restored when the deletion of SEN1990 was complemented. Our findings suggests that SEN1990 encodes a wHTH domain-containing transcriptional activator that modulates the transcription of oafB from the SPI-17, suggesting a crosstalk between these pathogenicity islands and a possible new role of ROD21 in the pathogenesis of Salmonella ser. Enteritidis. Overall design: Salmonella ser. Enteritidis P125109 Wild-type and ?SEN1990::frt strains were cultured overnight at 37°C in LB broth with agitation, and diluted in LB to OD600 0.01. The cultures were incubated at 37°C with agitation until OD600=0.6, at which samples were centrifugated at 8,000g for 8 min, the supernatant was discarded, and RNA was isolated using TRizol. Samples were further purified from salts using drop dialysis in MF-Millipore™ membranes (0.025µm pore diameter) against nuclease-free water for 4 h on ice. RNA-seq was performed as follows: Libraries were prepared using the Zymo-Seq RiboFree Total RNA Library Prep Kit and sequenced on an Illumina NovaSeq platform to a sequencing depth of at least 30 million read pairs (150 bp paired-end sequencing) per sample.