This study aimed to elucidate determinants of heat resistance in Escherichia coli by comparing the composition of membrane lipids, as well as gene expression, in heat-resistant E. coli AW1.7 and heat-sensitive E. coli GGG10 with or without heat shock. The survival of E. coli AW1.7 at late exponential phase was 100-fold higher than that of E. coli GGG10 after incubation at 60°C for 15 min. The cytoplasmic membrane of E. coli AW1.7 contained a higher proportion of saturated and cyclopropane fatty acids than that of E. coli GGG10. Microarray hybridization of cDNA libraries obtained from exponentially growing or heat-shocked cultures was performed to compare gene expression in these two strains. Expression of selected genes from different functional groups was quantified by quantitative PCR. DnaK and 30S and 50S ribosomal subunits were overexpressed in E. coli GGG10 relative to E. coli AW1.7 upon heat shock at 50°C, indicating improved ribosome stability. The outer membrane porin NmpC and several transport proteins were overexpressed in exponentially growing E. coli AW1.7. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of membrane properties confirmed that NmpC is present in the outer membrane of E. coli AW1.7 but not in that of E. coli GGG10. Expression of NmpC in E. coli GGG10 increased survival at 60°C 50-to 1,000-fold. In conclusion, the outer membrane porin NmpC contributes to heat resistance in E. coli AW1.7, but the heat resistance of this strain is dependent on additional factors, which likely include the composition of membrane lipids, as well as solute transport proteins.Escherichia coli is a common contaminant of the food supply. The majority of the strains of this species are not pathogenic; however, their relatively high resistance to environmental insults and the occurrence of virotypes with a low infectious dose, particularly enterohemorrhagic E. coli, make E. coli an organism of major concern in the production of minimally processed foods, particularly produce and fresh beef (11).Most strains of E. coli have a D 60 value (the duration of heat treatment at 60°C required to reduce the number of microorganisms to 1/10 of the initial value) of less than 1 min. However, the heat resistance of E. coli is highly variable among different strains and individual strains exhibit D 60 values of up to 6.5 min (8,21,25,37). The heat resistance of individual strains of E. coli relates to their ability adapt to heat stress by the homoviscous adaptation of the plasma membrane, as well as the synthesis of heat shock proteins (20, 43). The 32 -induced expression of heat shock proteins after sublethal thermal stress increases resistance to lethal heat treatment (43; for a review, see reference 6). Increased basal expression of the heat shock proteins DnaK, Lon, and ClpX was linked to the increased heat resistance of E. coli mutants LMM1010, LMM1020, and LMM 1030 (1, 21). The S -mediated general stress response additionally contributes to acid, heat, pressure, and salt resistance in E. coli (2,14,22,3...