Location: Produce Safety and Microbiology Research
Title: DNA Adenine Methylase, not the Pstl restriction-modification system, regulates virulence gene expression in Shiga toxin-producing Escherichia coliAuthor
Carter, Michelle | |
PHAM, ANTARES - US Department Of Agriculture (USDA) | |
Huynh, Steven | |
Parker, Craig | |
MILLER, AVALAN - US Department Of Agriculture (USDA) | |
He, Xiaohua | |
HU, BIN - Los Alamos National Research Laboratory | |
CHAIN, PATRICK S.G. - Los Alamos National Research Laboratory |
Submitted to: Food Microbiology
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 12/22/2020 Publication Date: 12/31/2020 Citation: Carter, M.Q., Pham, A., Huynh, S., Parker, C., Miller, A., He, X., Hu, B., Chain, P. 2020. DNA Adenine Methylase, not the Pstl restriction-modification system, regulates virulence gene expression in Shiga toxin-producing Escherichia coli. Food Microbiology. 96. Article 103722. https://doi.org/10.1016/j.fm.2020.103722. DOI: https://doi.org/10.1016/j.fm.2020.103722 Interpretive Summary: The genomes of Shiga toxin-producing Escherichia coli (STEC) are packed up with prophage or prophage-like DNA segments, which can be induced under certain conditions and transferred to other bacteria, a process known as horizontal gene transfer (HGT). HGT has played key roles in genome and virulence evolution of STEC since the genes encoding Shiga toxins (Stxs) are located on the prophage genomes. Recent studies suggest that phage mediated HGT not only leads to acquisition of virulence genes by host bacteria, but also impacts host bacteria fitness and pathogenicity via other factors encoded by the phage. In this study, we investigated the function of a DNA modification-Restriction system (PstI R-M) encoded by a Stx-converting prophage in the STEC O145:H28 strain linked to the 2010 lettuce-associated outbreak in the U.S. Our data revealed that, unlike the PstI R-M system in the STEC O104:H4 outbreak strain, the PstI R-M in STEC O145:H28 played a limited role in gene regulation; rather, it conferred STEC O145:H28 cells resistance to several antibiotics and fungicides that target to protein synthesis, energy metabolism, or membrane permeability. Furthermore, the PstI R-M system in STEC O145:H28 contributed to the maximum production of Shiga toxin following mitomycin C induction. In contrast, DNA adenine methylase (Dam), encoded by an E. coli core gene (not subject to HGT), played a key role in regulating virulence gene expression including the stx genes located on a propahge, the type III secretion systems genes located on a large pathogenicity island, and the enterohemolysin genes located on a virulence plasmid. Furthermore, Dam regulated a large number of genes associated with mobile genetic elements (MGEs) in STEC O145:H28, including the prophage genes related to host bacteria gene expression, metabolism, phage maturation, and host cell lysis, suggesting a co-evolution of MGEs with host bacteria. Repression of prophages by Dam in STEC supports the dam gene as a molecular target for STEC inactivation. Technical Abstract: We previously reported a distinct methylome between the two Shiga toxin-producing Escherichia coli (STEC) O145:H28 strains linked to the 2010 U.S. lettuce-associated outbreak (RM13514) and the 2007 Belgium ice cream-associated outbreak (RM13516), respectively. This difference was thought to be attributed to a prophage encoded type II restriction-modification system (PstI R-M) in RM13514. Here, we characterized this PstI R-M system in comparison to DNA adenine methylase (Dam), a highly conserved enzyme in gamma proteobacteria, by functional genomics. Deficiency in Dam led to a differential expression of over 1,000 genes in RM13514; whereas deficiency in the PstI R-M system only impacted a few genes transcriptionally. Dam regulated genes involved in diverse functions; whereas PstI R-M regulated genes mostly encoding transporters and adhesins. Furthermore, Dam regulated a large number of genes located on prophages, pathogenicity islands, and plasmids, including the Shiga toxin genes, type III secretion system (TTSS) genes, and enterohemolysin genes. Production of Stx2 in the dam mutant was significantly higher than that of RM13514, supporting a role of Dam in maintaining lysogeny of Stx2-converting prophage. However, following mitomycin C treatment, Stx2 in RM13514 was significantly higher than that of the dam or the PstI R-M deletion mutant, implying that both Dam and PstI R-M contributed to maximum Stx2 production. |