Identification of genes expressed in cultures of E. coli lysogens carrying the Shiga toxin-encoding prophage Φ24B

BMC Microbiology

Research Article

Marta Veses-Garcia¹  Alan J McCarthy¹  Heather E Allison¹  Laura M Riley²  Jeffrey D Hillman³  Martin Handfield³ 

[1] Microbiology Research Group, Institute of Integrative Biology, University of Liverpool, BioSciences Building, Crown Street, L69 7ZB, Liverpool, UK;Microbiology Research Group, Institute of Integrative Biology, University of Liverpool, BioSciences Building, Crown Street, L69 7ZB, Liverpool, UK;Program in Molecular Structure & Function, The Hospital for Sick Children, 555 University Avenue, M5G 1X8, Toronto, ON, Canada;Oragenics, 13700 Progress Blvd, 32615, Alachua, FL, USA;

关键词: Phage Genome;    Shiga Toxin;    Lytic Cycle;    Phage Gene;    EDTA Free Protease Inhibitor Cocktail;   

DOI  :  10.1186/1471-2180-12-42

 received in 2011-11-15, accepted in 2012-03-22,  发布年份 2012

来源: Springer

【 摘 要 】

BackgroundShigatoxigenic 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.ResultsLysogen 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.ConclusionPropagation 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.

【 授权许可】

Unknown   
© Riley et al; licensee BioMed Central Ltd. 2012. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.