Title: Peroxide resistance in Escherichia coli serotype O157:H7 biofilms is regulated by both RpoS dependent and independent mechanisms Authors
Submitted to: Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 11, 2012
Publication Date: June 14, 2012
Citation: Uhlich, G.A., Chen, C., Cottrell, B.J., Irwin, P.L., Phillips, J.G. 2012. Peroxide resistance in Escherichia coli serotype O157:H7 biofilms is regulated by both RpoS dependent and independent mechanisms. Microbiology. 158:2225-2234. Interpretive Summary: Escherichia coli O157:H7 and other Shiga toxin-producing E. coli (STEC) are important food-borne pathogens. In slaughter and food processing plants, sanitizers are used to eliminate or control food-borne pathogens. One of the mechanisms that sanitizers, such as hydrogen peroxide (H2O2), utilize to inactivate microbes is the production of oxygen free radicals. E. coli produces enzymes that inactivate H2O2 and other peroxides, including three enzymes encoded by the genes katG, katE, and ahpC. Many STEC encode an additional gene, katP. In a previous study, we characterized the role and regulation of all 4 genes in the resistance of E. coli O157:H7 to challenge with H2O2. However, in addition to peroxide inactivating enzymes, bacteria living on solid surfaces under nutrient-limited conditions also have the ability to form biofilms which provide a physical barrier against chemical agents. In this study we characterized the function and regulation of all 4 catalase/peroxidase genes in the protection of strain E. coli O157:H7 existing in 48- and 72-h biofilms. We confirmed that biofilm cells are more resistant than free living cells and showed that each of the four genes has a role in protecting biofilms against peroxide challenges. We also showed that the global gene regulator RpoS is essential for peroxide resistance in biofilms but a different regulator, OxyR, is not. In mature biofilms the katG and katE genes were the most protective in biofilms. This study is the first to show how the catalase/peroxidase genes function together with biofilm formation to protect E. coli O157:H7 from peroxides. The results of these studies will help researchers design new strategies for controlling food pathogens.
Technical Abstract: In many Escherichia coli serotype O157:H7 strains, defenses against peroxide damage include the peroxiredoxin AhpCF and three catalases: KatG (catalase-peroxidase), KatE (catalase), and the plasmid-encoded KatP (catalase/peroxidase). AhpC, KatG, and KatP are induced by OxyR /s70 in exponential phase, while KatE is regulated by RpoS during stationary growth. In a previous study we used mutant strains of ATCC 43895 (EDL933) with deletions of katG, ahpC, katE, and katP in all possible combinations to characterize peroxide resistance during both logarithmic and 18 to 24-hr growth in LB broth at 37 degrees C. In this study, we used triple deletion strains that isolated each catalase/peroxidase gene to investigate their role in the peroxide resistance of biofilm-forming variant 43895OR in 48- and 72-h biofilms. We also used qRT-PCR and translational lacZ fusions to study gene expression. Peroxide resistance was greater in biofilm cells than planktonic cells, and full resistance required rpoS but not oxyR. In 72-h biofilms, katG and katE were the major protective genes. None of the peroxidase/catalase genes were significantly induced by H2O2 in biofilms. katG, ahpC and katE peroxide protection had both rpoS-dependent and rpoS-independent components, but katP protection was independent of rpoS.