Enteropathogenic E. coli and enterohemorrhagic E. coli are food– and water–borne pathogens that are important causes of morbidity and mortality worldwide. These enteric pathogens contain a type III secretion system (T3SS) encoded by the Locus of Enterocyte Effacement (LEE) pathogenicity island, which is responsible for the hallmark histopathology known as the attaching and effacing (A/E) lesion. Expression of the LEE is controlled by a number of global transcriptional regulators and two of the most prominent are H–NS, a negative regulator, and Ler, the LEE–encoded master positive regulator. We investigated Ler and identified a second methionine as the native start residue. A trypsin proteolytic protection assay of Ler suggests the C–terminus is structured and NMR analysis of the Ler C–terminus revealed a structured protein not affected by magnesium cations. Size exclusion chromatography confirmed that Ler and its N–terminus form soluble aggregates. Another LEE–encoded regulator, the Multiple point controller (Mpc), has been reported to be important for LEE expression in EHEC. Our analysis demonstrated that Mpc did not have an effect on Ler–DNA–binding nor does it bind DNA itself and so its role in regulation is unclear. A paralogue of H-NS, H–NST, has been shown to affect the expression of H-NS regulon in E. coli K–12. We identified that H–NST positively affects the levels of LEE-encoded proteins independently of Ler and influences A/E lesion formation. We demonstrate that H-NST binds to regulatory regions LEE1 and LEE3, which is the first report of DNA–binding by H-NST. We identified that single and double amino acid substitutions (A16V, A16L, R60Q, and R60Q/R63Q) within H–NST negatively affect DNA binding and decrease LEE expression and A/E lesion formation. H–NST positively affects Ler binding to LEE regulatory regions in the presence of H–NS. Together, these studies provide new insights into the complex regulatory network that control expression of the LEE.