Characterizing RecA-Independent Induction of Shiga toxin2-Encoding Phages by EDTA

Background: The bacteriophage life cycle has an important role in Shiga toxin (Stx) expression. The induction of Shiga toxin-encoding phages (Stx phages) increases toxin production as a result of replication of the phage genome, and phage lysis of the host cell also provides a means of Stx toxin to exit the cell. Previous studies suggested that prophage induction might also occur in the absence of SOS response, independently of RecA. Methodology/Principal Findings: The influence of EDTA on RecA-independent Stx2 phage induction was assessed, in laboratory lysogens and in EHEC strains carrying Stx2 phages in their genome, by Real-Time PCR. RecA-independent mechanisms described for phage l induction (RcsA and DsrA) were not involved in Stx2 phage induction. In addition, mutations in the pathway for the stress response of the bacterial envelope to EDTA did not contribute to Stx2 phage induction. The effect of EDTA on Stx phage induction is due to its chelating properties, which was also confirmed by the use of citrate, another chelating agent. Our results indicate that EDTA affects Stx2 phage induction by disruption of the bacterial outer membrane due to chelation of Mg. In all the conditions evaluated, the pH value had a decisive role in Stx2 phage induction. Conclusions/Significance: Chelating agents, such as EDTA and citrate, induce Stx phages, which raises concerns due to their frequent use in food and pharmaceutical products. This study contributes to our understanding of the phenomenon of induction and release of Stx phages as an important factor in the pathogenicity of Shiga toxin-producing Escherichia coli (STEC) and in the emergence of new pathogenic strains. Citation: Imamovic L, Muniesa M (2012) Characterizing RecA-Independent Induction of Shiga toxin2-Encoding Phages by EDTA Treatment. PLoS ONE 7(2): e32393. doi:10.1371/journal.pone.0032393 Editor: Herman Tse, The University of Hong Kong, Hong Kong Received May 26, 2011; Accepted January 29, 2012; Published February 29, 2012 Copyright: 2012 Imamovic, Muniesa. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by the Generalitat de Catalunya (grant 2009SGR1043), by the Spanish Ministry of Education and Science (grant AGL200907576), and by the Xarxa de Referència en Biotecnologia (XeRBa). L. Imamovic is a recipient of a grant from the Spanish Ministry of Education and Science (grant FPI 20060054361). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected]