Enterohemorrhagic Escherichia coli (EHEC) is a virulent pathotype of E. coli that is associated with major outbreaks of hemorrhagic colitis and the life-threatening kidney disease hemolytic uremic syndrome. For successful host colonization and attachment to the intestinal mucosa, EHEC requires the locus of enterocyte effacement (LEE) pathogenicity island, which encodes a type III secretion system (TTSS) responsible for secreting and translocating effector proteins into host colonocytes. Regulation of the LEE is primarily directed through the first operon, LEE1, encoding the locus encoded regulator (Ler), and occurs through the direct and indirect action of several regulators. The 2006 U.S. spinach outbreak of E. coli O157:H7, characterized by unusually severe disease, has been attributed to a strain (TW14359) with enhanced pathogenic potential including elevated virulence gene expression, robust adherence, and the presence of novel virulence factors. Aim 1 of this dissertation proposes a mechanism for the unique virulence expression and adherence phenotype of this strain, and further expands the role for regulator RcsB in control of the E. coli locus of enterocyte effacement (LEE) pathogenicity island. Proteomic analysis of TW14359 revealed a virulence proteome consistent with previous transcriptome studies that included elevated levels of the LEE regulatory protein Ler and type III secretion system (T3SS) proteins, secreted T3SS effectors, and Shiga toxin 2. Basal levels of the LEE activator and Rcs phosphorelay response regulator, RcsB, were increased in strain TW14359 relative to O157:H7 strain Sakai. Deletion of rcsB eliminated inherent differences between these strains in ler expression, and in T3SS-dependent adherence. A reciprocating regulatory pathway involving RcsB and LEE-encoded activator GrlA was identified and predicted to coordinate LEE activation with repression of the flhDC flagellar regulator and motility. Overexpression of grlA was shown to increase RcsB levels, but did not alter expression from promoters driving rcsB transcription. Expression of rcsDB and RcsB was determined to increase in response to physiologic levels of bicarbonate, and bicarbonate-dependent stimulation of the LEE was shown to be dependent on an intact Rcs system and ler activator grvA. The results of this aim significantly broaden the role for RcsB in EHEC virulence regulation. The bicarbonate ion (HCO3-) has been shown to stimulate LEE gene transcription through the LEE1 promoter, and is predicted to serve as a physiologic signal for EHEC colonization. Results from the previous aim demonstrated that bicarbonate induction of the LEE is mediated through the Rcs phosphorelay, and is dependent upon an intact global regulator of virulence grvA gene. However, the direct mechanism through which RcsB-GrvA regulates ler, and the contribution of GrvA to the virulence of EHEC is unknown. In Aim 2, the RcsB-GrvA regulon of EHEC was determined by RNA sequencing, and the contributions of each to virulence and stress fitness was explored. A significant increase in transcription of the gad genes for extreme acid resistance was observed for both EHEC strains TW14359grvA and TW14359rcsBgrvA compared to TW14359, and corresponded with a significant increase in acid survival for TW14359grvA during exponential growth. Therefore, a model by which RcsB-GrvA coordinate LEE expression with acid resistance through GadE was proposed. Finally, the temporal regulation of both rcsDB and grvAB operons in response to bicarbonate was defined using single copy luxE chromosomal reporter fusions. Taken together, these results demonstrate the role of RcsB and GrvA to EHEC virulence, and reveal a novel role for GrvA in of extreme acid resistance and LEE gene expression and in EHEC. Finally, production of the ECP pilus has been demonstrated in enterohemorrhagic Escherichia coli O157:H7 (EHEC), and has been shown to be required for efficient adherence to epithelial cells during colonization. The first gene of the ecpRABCDE operon encodes a transcriptional regulator (EcpR) that positively regulates its own transcription, and promotes transcription and production of the downstream gene, ecpA, encoding the major ECP subunit EcpA. However, the distance between the ecpR and ecpA genes suggests the presence of regulatory elements that control ecpA directly. Therefore, it was hypothesized that an additional promoter was able to direct transcription of ecpA, independent of the promoter upstream of ecpR. To test this, promoter-lacZ transcriptional reporter fusions were created using the regions upstream of ecpR and ecpA to test for promoter activity, coupled with western blot analysis to detect EcpA in both wild-type and ecpR promoter mutant strains. In Aim 3, we showed that an additional promotable element, downstream of the EHEC O157:H7 strain TW14359 ecpR translational start site, is capable of driving transcription of ecpA, and that its activity is independent of an intact ecpR promoter. In addition, site-directed mutagenesis was used to characterize a TW14359 specific single nucleotide polymorphism within the predicted ecpA promoter region. Overproduction of EcpR was observed to increase cytosolic RcsB and Tir, indicating that ecp production is able to stimulate the LEE, and that the ecpA promoter polymorphism may contribute to intrinsically increased rcsB transcription in TW14359. Taken together, the results, and those obtained in Aims 1 and 2, expand the model for regulation of the ecp operon in EHEC O157:H7 strain TW14359, and broaden the model for EcpR and RcsB in the coordinate regulation of E. coli common pilus and type III secretion.