title: Identification of genes expressed in cultures of E. coli lysogens carrying the Shiga toxin-encoding prophage Φ24B.
abstract: BACKGROUND
  Shigatoxigenic E. coli are a global and emerging health concern. Shiga toxin, Stx, is encoded on the genome of temperate, lambdoid Stx phages. Genes essential for phage maintenance and replication are encoded on approximately 50% of the genome, while most of the remaining genes are of unknown function nor is it known if these annotated hypothetical genes are even expressed. It is hypothesized that many of the latter have been maintained due to positive selection pressure, and that some, expressed in the lysogen host, have a role in pathogenicity. This study used Change Mediated Antigen Technology (CMAT)™ and 2D-PAGE, in combination with RT-qPCR, to identify Stx phage genes that are expressed in E. coli during the lysogenic cycle.
  
  
  RESULTS
  Lysogen cultures propagated for 5-6 hours produced a high cell density with a low proportion of spontaneous prophage induction events. The expression of 26 phage genes was detected in these cultures by differential 2D-PAGE of expressed proteins and CMAT. Detailed analyses of 10 of these genes revealed that three were unequivocally expressed in the lysogen, two expressed from a known lysogenic cycle promoter and one uncoupled from the phage regulatory network.
  
  
  CONCLUSIONS
  Propagation of a lysogen culture in which no cells at all are undergoing spontaneous lysis is impossible. To overcome this, RT-qPCR was used to determine gene expression profiles associated with the growth phase of lysogens. This enabled the definitive identification of three lambdoid Stx phage genes that are expressed in the lysogen and seven that are expressed during lysis. Conservation of these genes in this phage genome, and other Stx phages where they have been identified as present, indicates their importance in the phage/lysogen life cycle, with possible implications for the biology and pathogenicity of the bacterial host.
authors.affiliation: Microbiology Research Group, Institute of Integrative Biology, University of Liverpool, BioSciences Building, Crown Street, Liverpool L69 7ZB, UK.
authors.forename: Laura M
authors.initials: LM
authors.lastname: Riley
authors.forename: Marta
authors.initials: M
authors.lastname: Veses-Garcia
authors.forename: Jeffrey D
authors.initials: JD
authors.lastname: Hillman
authors.forename: Martin
authors.initials: M
authors.lastname: Handfield
authors.forename: Alan J
authors.initials: AJ
authors.lastname: McCarthy
authors.forename: Heather E
authors.initials: HE
authors.lastname: Allison
mesh_terms: D010582:Bacteriophage lambda / Q000235:genetics* / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D005814:Genes, Viral; D008242:Lysogeny; D022621:Shiga Toxin / Q000235:genetics / Q000378:metabolism*; D054323:Shiga-Toxigenic Escherichia coli / Q000254:growth & development / Q000821:virology*; D059467:Transcriptome
chemical_list: D022621:Shiga Toxin