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Dam and Stable 933W Lysogeny

Reference:Murphy, K.C.,, Ritchie, J.M., Waldor, M.K., LÝbner-Olesen, A. and M.G. Marinus (2008) Dam methyltransferase is required for stable lysogeny of the Shiga Toxin (Stx2)-encoding bacteriophage 933W of enterohemorrhagic Escherichia coli O157:H7. J. Bacteriol. 190, 438-441.

Shiga toxin 2 (Stx2), one of the principal virulence factors of enterhemorrhagic E. coli is encoded by 933W, a lambda-like prophage. 933W prophage induction contributes to Stx2 production and here we provide evidence that Dam methyltransferase is essential for maintenance of 933W lysogeny. Our findings are consistent with the idea that the 933W prophage has a relatively low threshold for induction, which may promote Stx2 production during infection.





Enterohemorrhagic E. coli Pedestal Formation

Reference: Campellone, K.G., Roe,  A.J., Løbner-Olesen, A., Murphy, K.C., Magoun, L., Brady, M.J., Donohue-Rolfe, A.J., Tzipori, S., Gally, D.L., Leong, J.M. and Marinus, M. G. (2007) Increased adherence and actin pedestal formation by dam-deficient enterohemorrhagic Escherichia coli O157:H7. Molec. Microbiol. 63, 1468-1481.

Enterohemorrhagic Escherichia coli (EHEC) are highly-infectious pathogens capable of causing severe diarrheal illnesses. As a critical step during their colonization, EHEC adhere intimately to intestinal epithelial cells and generate F-actin ‘pedestal’ structures that elevate them above surrounding cell surfaces. Intimate adhesion and pedestal formation result from delivery of the EHEC type III secretion system (TTSS) effector proteins Tir and EspFU into the host cell and expression of the bacterial outer membrane adhesin, intimin. To investigate a role for DNA methylation during the regulation of adhesion and pedestal formation in EHEC, we deleted the dam (DNA adenine methyltransferase) gene from EHEC O157:H7 and demonstrate that this mutation results in increased interactions with cultured host cells. EHECΔdam exhibits dramatically elevated levels of adherence and pedestal formation when compared to wild type EHEC, and expresses significantly higher protein levels of intimin, Tir and EspFU. Analyses of GFP fusions, Northern blotting, RT-PCR, and microarray experiments indicate that the abundance of Tir in the dam mutant is not due to increased transcription levels, raising the possibility that Dam methylation can indirectly control protein expression by a post-transcriptional mechanism. In contrast to other dam-deficient pathogens, EHECΔdam was capable of robust intestinal colonization of experimentally infected animals.




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