Enterohemorrhagic Escherichia coli (EHEC) O157:H7 responds to the host-produced epinephrine and norepinephrine, and bacterially produced autoinducer 3 (AI-3), through two-component systems. Further integration of multiple regulatory signaling networks, involving regulators such as the LysR-type transcriptional regulator (LTTR) QseA, promotes effective regulation of virulence factors. These include the production of flagella, a phage-encoded Shiga toxin, and genes within the locus of enterocyte effacement (LEE) responsible for attaching and effacing (AE) lesion formation. Here, we describe a new member of this signaling cascade, an LTTR heretofore renamed QseD (quorum-sensing E. coli regulator D). QseD is present in all enterobacteria but exists almost exclusively in O157:H7 isolates as a helix-turn-helix (HTH) truncated isoform. This "short" isoform (sQseD) is still able to regulate gene expression through a different mechanism than the full-length K-12 E. coli "long" QseD isoform (lQseD). The EHEC ⌬qseD mutant exhibits increased expression of all LEE operons and deregulation of AE lesion formation. The loss of qseD in EHEC does not affect motility, but the K-12 ⌬qseD mutant is hypermotile. While the lQseD directly binds to the ler promoter, encoding the LEE master regulator, to repress LEE transcription, the sQseD isoform does not. LTTRs bind to DNA as tetramers, and these data suggest that sQseD regulates ler by forming heterotetramers with another LTTR. The LTTRs known to regulate LEE transcription, QseA and LrhA, do not interact with sQseD, suggesting that sQseD acts as a dominant-negative partner with a yet-unidentified LTTR.Enterohemorrhagic Escherichia coli (EHEC) is the causative agent of outbreaks of hemorrhagic colitis and hemolytic uremic syndrome (HUS) throughout the world. Of the multiple pathogenic serotypes of clinical importance, O157:H7, a serotype that is believed to have evolved recently from an O55:H7 atypical enteropathogenic E. coli (aEPEC) strain, is by far the most prevalent and virulent (18,34,40). EHEC strains are part of a larger group of enteric pathogens that includes enteropathogenic E. coli (EPEC), a rabbit EPEC strain, and Citrobacter rodentium, all of which are able to cause attaching and effacing (AE) lesions on intestinal epithelial cells (80).The genes necessary for the formation of these characteristic AE lesions are chromosomally located within the locus of enterocyte effacement (LEE) pathogenicity island (PAI) (49). The LEE is composed of 41 genes, including the LEE-encoded regulator gene (ler) that activates transcription of all LEE genes (50). The majority of the LEE genes are arranged into five major operons that encode both structural proteins that form a type three secretion system (TTSS) and several of the secreted effectors, such as the translocated intimin receptor (Tir), EspH, and Map, which are translocated through the TTSS into host cells (15,20,32,36). Once translocated, Tir embeds itself in the host membrane, where its extracellular domain serves as a docking p...