Genetic Regulation of the Locus of Heat Resistance in Escherichia coli

Abstract

During food processing, Escherichia coli are exposed to stress from oxidation, osmolarity and temperature, and have developed mechanisms to survive. A highly heat resistant strain of E. coli AW1.7 was isolated from a beef carcass after thermal processing and it has a genomic island called the locus of heat resistance (LHR) that has 16 predicted open reading frames. The LHR has been reported in E. coli isolated from raw cheese milk and treated wastewater and contributes to high pressure and chlorine resistance. Most studies on the LHR has focused on genetic function or its role on the survival under different types of stress, but there has been limited research on its genetic regulation. Previous studies predicted that EvgA is a regulator of the LHR because it induces yfdX expression and the LHR contains two hypothetical proteins from the YfdX family. The current research aims to investigate the regulatory mechanisms of the LHR as its expression of genes in response to different stressors remains unclear. The objectives were to evaluate EvgA as a regulator of the LHR; investigate the presence of additional promoters using in-silico methods and validate their role using gene expression; and to examine the induction of promoters using stress. The constructed evgAS-pRK complement was not able to restore phenotype of the EvgA deficient Keio strain, E. coli MG1655 ΔevgA. Further investigation found that the pLHR plasmid was absent from E. coli MG1655 ΔevgA (pLHR), explaining its loss of heat resistance. CNNPromoter was used to predict six promoter sequences regulated by Crp, OmpR, PurR, FadR, RpoD or OxyR within the LHR. Overexpression of regulatory proteins using plasmids from the ASKA library and gene expression using RT-qPCR of genes downstream of the predicted promoters were used to determine promoter activity. EvgA, OxyR, and RpoD affected the expression of LHR genes. Crp, FadR, PurR, and OmpR had minor effects on gene expression. When the OxyR regulated promoter was induced by chlorine or hydrogen peroxide, expression of trx increased, supporting that OxyR regulates its respective promoter. In conclusion, there are regulatory sites within the LHR that may account for the differential expression of genes in response to stress.