Secretion of effector proteins in Enteropathogeneic Escherichia coli (EPEC) and Enterohemorrhagic Escherichia coli (EHEC) is mediated by a specialized type III secretion system, components of which are encoded in the LEE operons 1 to 5. H-NS, a global repressor in E. coli, silences the expression of LEE operons. Ler, a master regulator in LEE operons, shares 24% amnio acid identity and 44% amino acid similarity to H-NS. Interestingly, rather than a gene silencer, its main role has been characterized as an antagonizing protein that relieves H-NS-mediated transcriptional silencing. In the previous study we reported molecular mechanism for the repression of LEE5 promoter in EPEC and EHEC by H-NS as a protein interaction between upstream DNA-bound H-NS and the aCTD of promoter-bound RNA polymerase. The mechanism underlying Ler-mediated alleviation of the genes repression by H-NS is largely unknown. We examined regulatory effect of these proteins on LEE5p activity using various in vitro tools. Our results revealed that binding affinity of Ler to the LEE5p DNA is about 40 folds greater than that of HNS as determined by surface plasmon resonance. We verified that Ler binding removed H-NS bound to the same stretch of DNA on LEE5 promoter resulting in a derepression. (C) 2016 Elsevier Inc. All rights reserved.