Functional Analysis of ycfR and ycfQ in Escherichia coli O157:H7 Linked to Outbreaks of Illness Associated with Fresh Produce

Abstract

Fresh produce has been associated with multiple outbreaks of illness caused by Escherichia coli O157:H7. The mechanism of E. coli O157:H7 survival through postharvest processing of fresh produce needs to be understood to help develop more effective interventions. In our recent transcriptomic study of strain Sakai, an isolate from the 1996 sprout outbreak in Japan, and strain TW14359, an isolate from the 2006 spinach outbreak in the United States, we showed that ycfR was the most significantly upregulated gene in response to chlorine-based oxidative stress. YcfR is known to be a multiple stress resistance protein and a biofilm regulator in E. coli K-12 strains; however, its role in the pathogenic E. coli O157:H7 has not been clearly defined. In this study, ycfR was replaced with a chloramphenicol resistance cassette oriented in two different directions to construct polar and nonpolar ycfR::cat mutants of Sakai and TW14359. Chlorine resistance and survival on spinach leaf surfaces were assessed in the wild-type strains and the ycfR mutants. Both polar and nonpolar ycfR mutants of Sakai showed significantly less chlorine resistance than their parent strain. In contrast, deletion of ycfR in TW14359 did not change chlorine resistance, indicating that ycfR in these two outbreak-related E. coli O157:H7 strains may function differently. In addition, after a 24-h incubation on spinach leaves in a sublethal concentration of chlorine, the Sakai nonpolar ycfR mutant exhibited lower survival compared to the wild type. The results suggest a role for ycfR in survival of Sakai during chlorine exposure. We also found that the upstream ycfQ, which is annotated as a DNA-binding regulator, acted as a repressor of ycfR. These findings suggest that gene regulation may be a mechanism by which E. coli O157:H7 strain Sakai could survive in the postharvest processing environment.