APEC IMT5155 aatA : genomic locus and predicted protein structure. A: Scheme of the genomic locus of aatA in IMT5155. The open reading frame (ORF) of aatA is indicated as grey arrow. Black bars indicate PCR fragments amplified using oligonucleotides marked as black arrows. The size of each fragment and the ORF are given in brackets. B: Scheme of the predicted protein AatA. The 3,498 bp ORF results in the 124-kDa A PEC a ut otransporter adhesin A. Sequence analyses revealed the given domain structure. At the N-terminus a signal peptide (SP) is predicted which probably enables the sec machinery to secrete AatA across the cytoplasmic membrane. The autotransporter repeat (ATr) is found in many AT adhesins and proteins, which are predicted as AT adhesins. The alignment below the protein structure shows conserved amino acid (aa) residues within one AT repeat. C-terminal of the AT repeat lies the predicted functional passenger domain found in AT adhesins (PD). The AT-adhesin-typical translocation domain (TD) resides at the C-terminus of the protein. C: Scheme of fusion protein AatAF. Using oligonucleotides B11-for and B11-rev the 1,222 bp fragment aatA_1222, comprising the region for the AT repeat and the functional PD was amplified by PCR and cloned into pET32a(+) for expression. The 64-kDa fusion protein AatAF contains an enterokinase recognition site (EK), an S tag, a thrombin site (T), a His6 tag and a thioredoxin tag (Trx) fused to the N-terminus of the adhesin peptide to enhance protein solubility and to simplify protein purification.