Escherichia coli O104:H4 (E. coli O104:H4), which caused a massive outbreak of acute gastroenteritis and hemolytic uremic syndrome in 2011, carries an aggregative adherence fimbriae I (AAF/I) encoding virulence plasmid, pAA. The importance of pAA in host-pathogen interaction and disease severity has been demonstrated, however, not much is known about its transcriptional organization and gene regulation. Here, we analyzed the pAA primary transcriptome using differential RNA sequencing, which allows for the high-throughput mapping of transcription start site (TSS) and non-coding RNA candidates. We identified 248 TSS candidates in the 74-kb pAA and only 21% of them could be assigned as TSS of annotated genes. We detected TSS for the majority of pAA-encoded virulence factors. Interestingly, we mapped TSS, which could allow for the transcriptional uncoupling of the AAF/I operon, and potentially regulatory antisense RNA candidates against the genes encoding dispersin and the serine protease SepA. Moreover, a computational search for transcription factor binding sites suggested for AggR-mediated activation of SepA expression, which was additionally experimentally validated. This work advances our understanding of the molecular basis of E. coli O104:H4 pathogenicity and provides a valuable resource for further characterization of pAA virulence gene regulation.

The airways of the majority of adolescent cystic fibrosis (CF) patients are persistently colonized or infected by Staphylococcus aureus. Using whole genome sequencing, we studied the evolutionary traits within a S. aureus population in the airways of a CF patient hypothesizing that horizontal gene transfer (HGT) and inter-bacterial interaction play a major role in adaptation during long-term persistence. Whole genome sequencing of 21 S. aureus isolates spanning 13 years resulted in seven lineages defined by the spa types t012, t021, t331, t338, t364, t056, and t2351. Of these, the successfully persisting lineages t012 and t021 were closely related suggesting the evolution of t021 from t012, which was further corroborated by a nearly identical, syntenic set of mobile genetic elements. During transformation from t012 to t021, an increase of genomic changes including HGT from other S. aureus lineages was detected. In summary, our in vivo data enabled us to conceptualize an evolutionary model showing the impact of HGT and inter-bacterial interaction on bacterial long-term adaptation to the human host during CF.

Enterohemorrhagic Escherichia coli (EHEC) can cause severe diarrheic in humans. To improve therapy options, a better understanding of EHEC pathogenicity is essential. The genetic manipulation of EHEC with classical one-step methods, such as the transient overexpression of the phage lambda (λ) Red functions, is not very efficient. Here, we provide a robust and reliable method for increasing recombineering efficiency in EHEC based on the transient coexpression of recX together with gam, beta, and exo. We demonstrate that the genetic manipulation is 3–4 times more efficient in EHEC O157:H7 EDL933 Δstx1/2 with our method when compared to the overexpression of the λ Red functions alone. Both recombineering systems demonstrated similar efficiencies in Escherichia coli K-12 MG1655. Coexpression of recX did not enhance the Gam-mediated inhibition of sparfloxacin-mediated SOS response. Therefore, the additional inhibition of the RecFOR pathway rather than a stronger inhibition of the RecBCD pathway of SOS response induction might have resulted in the increased recombineering efficiency by indirectly blocking phage induction. Even though additional experiments are required to unravel the precise mechanistic details of the improved recombineering efficiency, we recommend the use of our method for the robust genetic manipulation of EHEC and other prophage-carrying E. coli isolates.

Background: Escherichia coli ( E. coli ) O104:H4 caused the 2011 enterohemorrhagic E. coli (EHEC) outbreak in Germany, which remains the outbreak with the highest incidence of hemolytic uremic syndrome worldwide. We recently identified an E. coli O104:H4 isolate carrying a single nucleotide polymorphism in the start codon (ATG > ATA) of rpoS , which encodes the alternative sigma factor RpoS, resulting in reduced RpoS levels and enhanced virulence gene expression. Methods and results: Gene set enrichment analysis further revealed that the rpoS ATG > ATA mutation was primarily associated with activation of numerous metabolic pathways and repression of carbon source utilization-related transporter and transcription factor genes. Consistently, BIOLOG phenotype microarrays showed that E. coli O104:H4 rpoS ATG > ATA assimilated amino acids and organic acids (TCA cycle substrates) more efficiently, whereas the wild type strain displayed stronger metabolic respiration with sugars and sugar derivatives, including constituents of the mucus. Deletion of rpoS (Δ rpoS ) in E. coli O104:H4 Δ stx2 resulted in a carbon source utilization profile similar to the one of rpoS ATG > ATA, as well as in enhanced growth in minimal medium supplemented with amino acids and reduced one with sugars. Moreover, co-culture experiments with E. coli O104:H4 Δ stx2 and E. coli O104:H4 Δ stx2 Δ rpoS revealed a strong competitive advantage of the Δ rpoS strain with the tested amino acids; however, no advantage of the rpoS -intact strain was observed with sugars. Conclusion: Our findings elucidate the impact of RpoS on global gene expression and carbon source utilization in E. coli O104:H4, further underscoring its role as a central regulator in pathogenic bacteria.