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The physiological and molecular responses of enterotoxigenic Escherichia coli K88 strain Bd 1107/7508 during growth in piglet ileal mucus and lipids extracted from mucus were studied in terms of growth rate, protein expression, and rate of heat production. E. coli K88 multiplied at maximum speed in mucus and in lipids extracted from mucus. By two-dimensional gel electrophoresis of [35S]methionine-labelled cells, it was demonstrated that the synthesis of a subclass of 13 proteins was changed at least fourfold during exponential growth in mucus compared with growth in M9 minimal medium. Ten of these proteins were repressed, while three were induced, and one of the induced proteins was identified as heat shock protein GroEL. Furthermore, two-dimensional analysis of E. coli K88 cells grown on lipids extracted from mucus revealed a set of lipid utilization-associated proteins. None of these was induced fourfold during exponential growth in mucus. Microcalorimetric measurements (monitoring the rate of heat production) of E. coli K88 grown in mucus indicated metabolic shifts in the stationary phase, in which five of the lipid utilization-associated proteins were expressed at a higher level. An isogenic E. coli K88 fadAB mutant deficient in fatty acid degradation genes grew as well as the wild type on mucus and mucus lipids. The heat production rate curve of the mutant grown in mucus differed from that of the wild type only during the stationary phase. From these results it was concluded that protein expression is influenced when E. coli K88 is grown in piglet ileal mucus rather than in M9 minimal medium. Lipids extracted from ileal mucus can serve as a substrate for E. coli K88 but appear not to be utilized during exponential growth in mucus. Stationary-phase cells metabolize fatty acids; however, the functional purpose of this is unclear.