title: Comparison of strand-specific transcriptomes of enterohemorrhagic Escherichia coli O157:H7 EDL933 (EHEC) under eleven different environmental conditions including radish sprouts and cattle feces.
abstract: BACKGROUND
  Multiple infection sources for enterohemorrhagic Escherichia coli O157:H7 (EHEC) are known, including animal products, fruit and vegetables. The ecology of this pathogen outside its human host is largely unknown and one third of its annotated genes are still hypothetical. To identify genetic determinants expressed under a variety of environmental factors, we applied strand-specific RNA-sequencing, comparing the SOLiD and Illumina systems.
  
  
  RESULTS
  Transcriptomes of EHEC were sequenced under 11 different biotic and abiotic conditions: LB medium at pH4, pH7, pH9, or at 15°C; LB with nitrite or trimethoprim-sulfamethoxazole; LB-agar surface, M9 minimal medium, spinach leaf juice, surface of living radish sprouts, and cattle feces. Of 5379 annotated genes in strain EDL933 (genome and plasmid), a surprising minority of only 144 had null sequencing reads under all conditions. We therefore developed a statistical method to distinguish weakly transcribed genes from background transcription. We find that 96% of all genes and 91.5% of the hypothetical genes exhibit a significant transcriptional signal under at least one condition. Comparing SOLiD and Illumina systems, we find a high correlation between both approaches for fold-changes of the induced or repressed genes. The pathogenicity island LEE showed highest transcriptional activity in LB medium, minimal medium, and after treatment with antibiotics. Unique sets of genes, including many hypothetical genes, are highly up-regulated on radish sprouts, cattle feces, or in the presence of antibiotics. Furthermore, we observed induction of the shiga-toxin carrying phages by antibiotics and confirmed active biofilm related genes on radish sprouts, in cattle feces, and on agar plates.
  
  
  CONCLUSIONS
  Since only a minority of genes (2.7%) were not active under any condition tested (null reads), we suggest that the assumption of significant genome over-annotations is wrong. Environmental transcriptomics uncovered hitherto unknown gene functions and unique regulatory patterns in EHEC. For instance, the environmental function of azoR had been elusive, but this gene is highly active on radish sprouts. Thus, NGS-transcriptomics is an appropriate technique to propose new roles of hypothetical genes and to guide future research.
authors.forename: Richard
authors.initials: R
authors.lastname: Landstorfer
authors.forename: Svenja
authors.initials: S
authors.lastname: Simon
authors.forename: Steffen
authors.initials: S
authors.lastname: Schober
authors.forename: Daniel
authors.initials: D
authors.lastname: Keim
authors.forename: Siegfried
authors.initials: S
authors.lastname: Scherer
authors.affiliation: Lehrstuhl für Mikrobielle Ökologie, Wissenschaftszentrum Weihenstephan, Technische Universität München, Weihenstephaner Berg 3, D-85350 Freising, Germany. neuhaus@wzw.tum.de.
authors.forename: Klaus
authors.initials: K
authors.lastname: Neuhaus
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002417:Cattle; D019453:Escherichia coli O157 / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics; D005243:Feces / Q000382:microbiology*; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D006863:Hydrogen-Ion Concentration; D031224:Raphanus / Q000235:genetics / Q000378:metabolism / Q000382:microbiology*; D017423:Sequence Analysis, RNA; D059467:Transcriptome / Q000187:drug effects; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D029968:Escherichia coli Proteins; D037521:Virulence Factors