A novel chromosomal locus of enteropathogenic Escherichia coli (EPEC), which encodes a bfpT-regulated chaperone-like protein, TrcA, involved in microcolony formation by EPEC

The bfpTVW operon, also known as the per operon, of enteropathogenic Escher ichia coli (EPEC) is required for the transcriptional activation of the bfp operon, which encodes the major subunit and assembly machinery of bundle-f orming pill (BFP). An immobilized T7-tagged BfpT fusion protein that binds specifically to upstream promoter sequences of bfpA and eae was used to 'fi sh out' from a promoter library other EPEC chromosomal fragments that are b ound by the BfpT protein. After screening for promoters exhibiting bfpTVW-d ependent expression, one was identified that was positively regulated by bf pTVW and that is not present in the chromosomes of two non-virulent E. coli laboratory strains, DH5 alpha and HB101. Further analysis of this positive ly regulated promoter in EPEC showed that it resided within a 4.9 kb sequen ce that is not present in E. coli K12. This locus, located downstream of th e potB gene, was found to contain four open reading frames (ORFs): bfpTVW-a ctivated promoter was localized upstream of ORF1. An ORF1 knockout mutant p roduced less of the BFP structural subunit (BfpA) and formed smaller than n ormal adherent microcolonies on cultured epithelial cells; however, this mu tation did not affect bfp transcription. An ORF1-His6 fusion protein specif ically bound the preprocessed and mature forms of the BfpA protein and thus appears to stabilize the former within the cytoplasmic compartment. ORF1 t herefore is a newly isolated EPEC chromosomal gene that encodes a chaperone -like protein involved in the production of BFP. Hence, ORF1 was designated trcA (bfpT-regulated chaperone-like protein gene). The TrcA protein also s pecifically bound 39 kDa and 90 kDa proteins that are expressed by EPEC but not by E. coli K12. The 90 kDa protein was revealed to be intimin, a prote in product of the eae gene, which is required for the EPEC attaching/effaci ng phenotype, suggesting a direct interaction of TrcA with intimin in the c ytoplasmic compartment.