Thermoregulation of the Escherichia coli O157:H7 pO157 ecf operon and lipid A myristoyl transferase activity involves intrinsically curved DNA

Abstract

Escherichia coli O157:H7 survives in diverse environments from the ruminant gastrointestinal tract to cool nutrient-dilute water. We hypothesized that the gene regulation required for this flexibility includes intrinsically curved DNA that responds to environmental changes. Three intrinsically curved DNAs were cloned from the E. coli O157:H7 virulence plasmid (pO157), sequenced 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 degrees C, six partially phased adenine:thymine tracts and transcriptional activation. BNT2::lacZ operon fusions showed that BNT2 activated transcription at 24 degrees C compared to 37 degrees C and was partially repressed by a bacterial nucleoid-associated protein H-NS. BNT2 regulated the E. coli attaching and effacing gene-positive conserved fragments 1-4 (ecf1-4) that are conserved in Shiga toxin-producing E. coli associated with human disease. Experimental analyses showed that ecf1-4 formed an operon. ecf1, 2 and 3 encoded putative proteins associated with bacterial surface polysaccharide biosynthesis and invasion and 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.