Characterization of the Escherichia coli O157:H7 pO157 ecf operon

Escherichia coli O157:H7 survives and persists well in diverse environments from the human and bovine gastrointestinal tract (GIT) to nutrient dilute farm water troughs. It was hypothesized that the gene regulation required for this flexibility includes intrinsically curved DNA that responds to environmental changes. In this study, the four genes regulated by intrinsically curved DNA were identified and their biological significance was examined in vivo and in vitro. Three intrinsically curved DNAs were isolated from the E. coli O157:H7 virulence plasmid (pO157) and designated Bent 1 through Bent 3 (BNT1, BNT2, and BNT3). Compared to BNT1 and BNT3, BNT2 had characteristics typical of intrinsically curved DNA including electrophoretic gel retardation at 4°C, six partially phased adenine:thymine tracts, and transcriptional activation. BNT2::lacZ operon fusions showed that BNT2 activated transcription at 24°C compared to 37°C and was partially repressed by a bacterial nucleoid-associated protein H-NS. BNT2 regulated the E&barbelow;. coli attaching and effacing gene-positive c&barbelow;onserved f&barbelow;ragments 1 to 4 (ecf1 to 4) that are conserved in Shiga toxin-producing E. coli associated with human disease. As an operon, ecf1 and 3 encoded proteins associated with bacterial surface polysaccharide biosynthesis and invasion and ecf2 and 4 encoded enzymes associated with the LPS inner core and lipid A modification. Genetic analyses showed that ecf4 complemented a chromosomal deletion of lpxM encoding lipid A myristoyl transferase. Mass spectrometric analysis of lipid A from ecf and lpxM single and double mutants showed that myristoylation was altered at lower temperature. Further analyses of these mutants in vivo and in vitro showed that the double mutant did not survive passage through the bovine GIT and did not persist well in farm water troughs. Also, these mutations altered the lipid A and membrane fatty acid composition, increased bacterial susceptibility to a broad spectrum of antibiotics and detergents, and impaired bacterial motility. Electron microscopic analyses showed changes in bacterial membrane structure. This is the first demonstration that the pO157-encoded genes affect E. coli O157:117 survival and persistence in in vivo and ex vivo environments and implies the role of the pO157 for bacterial survival and persistence in these environments.