Project description:We performed a high-throughput mapping of the 5’ end transcriptome of the pAA plasmid of the clinical Escherichia coli O104:H4 (E. coli O104:H4) isolate LB226692. We employed differential RNA-sequencing (dRNA-seq), a terminator exonuclease (TEX)-based RNA-seq approach allowing for the discrimination of primary and processed transcripts. This method has proven to be a powerful tool for the mapping of transcription start sites (TSS) and detection of non-coding RNAs (ncRNAs) in bacteria. We catalogued pAA-associated TSS and processing sites on a plasmid-wide scale and performed a detailed analysis of the primary transcriptome focusing on pAA virulence gene expression.
Project description:In 2011, in Germany, Escherichia coli O104:H4 caused the enterohemorrhagic E. coli (EHEC) outbreak with the highest incidence rate of hemolytic uremic syndrome. This pathogen carries an exceptionally potent combination of EHEC- and enteroaggregative E. coli (EAEC)-specific virulence factors. Here, we identified an E. coli O104:H4 isolate that carried a single nucleotide polymorphism (SNP) in the start codon (ATG>ATA) of rpoS, encoding the alternative sigma factor S. The rpoS ATG>ATA SNP was associated with enhanced EAEC-specific virulence gene expression. Deletion of rpoS in E. coli O104:H4 Dstx2 and typical EAEC resulted in a similar effect. Both rpoS ATG>ATA and DrpoS strains exhibited stronger virulence-related phenotypes in comparison to wild type. Using promoter-reporter gene fusions, we demonstrated that wild-type RpoS repressed aggR, encoding the main regulator of EAEC virulence. In summary, our work demonstrates that RpoS acts as a global repressor of E. coli O104:H4 virulence, primarily through an AggR-dependent mechanism.
