B-186. Transcriptional Analysis of the ecp Operon of Attaching and Effacing Escherichia coli

V. I. Martínez-Santos1, A. L. Erdem2, J. A. Girón2, J. L. Puente1;
1Instituto de Biotecnología, U.N.A.M., Cuernavaca, MEXICO, 2Univ. of Arizona, Tucson, AZ.

Enteropathogenic and Enterohemorrhagic E. coli are clinically important pathogens that colonize the small gut and colon, respectively. EPEC is a mayor cause of infantile diarrhea affecting principally children under 6 months in developing countries, while EHEC is the causative agent of hemorrhagic colitis and the often-lethal hemolytic uremic syndrome. These bacteria adhere to the intestinal epithelial surface and produce a histopathology known as the attaching and effacing lesion (A/E). Bacterial adherence can be mediated by polymeric fibers called “pili” or “fimbriae”. Recently, we have shown that the E. coli common pili (ECP), also called Mat, are expressed by several pathogenic and non-pathogenic E. coli strains. The EcpA fimbrial subunit is encoded by the second gene of the ecp operon, which is preceded by the gene coding for EcpR, which is proposed to code for a regulatory protein that contains a C-terminal HTH DNA-binding motif similar to the LuxR family of transcriptional regulators. In this study, we analyzed the transcriptional regulation of the ecp operon and the role of EcpR as a transcriptional regulator in EHEC and EPEC. Immunofluorescence microscopy revealed that an ecpR mutant adhere less efficiently to cultured epithelial cell lines than wild type EHEC and the complemented strain. Consistently, EcpA could not be detected by western blot in the ecpR mutant and was much less abundant in this strain as shown by flow cytometry. The ecp operon is transcribed from a putative sigma 70 promoter located upstream of ecpR. A deletion analysis of the ecp regulatory region using cat transcriptional fusions revealed that EcpR autoregulates itself positively by acting on a DNA motif located between positions -150 and -200 upstream of the transcriptional start site. Also, we identified a regulatory element located within the ecpR-ecpA intergenic region that modulates negatively the transcription of ecpA and seems to account, at least in part, for the differential regulation observed for EcpR and EcpA synthesis. Expression of ECP was also shown to be negatively and positively regulated by the global regulators H-NS and IHF, respectively.