The N-terminal part of the hypothetical protein (Figure 5, blue-purple area) is predicted to adopt a structure similar to the DNA-binding domains of the PhoB transcription factor. The characteristic HTH motif is a common feature of transcription factors. Although the PSPPH_2539 ORF is annotated in the NCBI as a LuxR-type of transcription regulator, the choice of the DNA-binding domain of PhoB as a structural template indicates that PSPPH_2539 probably has an α-/β- doubly wound fold (distinguished by the presence of a C-terminal β-strand

hairpin unit that packs Angiogenesis inhibitor against the shallow cleft of the partially open tri-helical HTH core) motif. Transcription factors are usually multidomain proteins, thus the assignment of PSPPH_2539 as a LuxR-type transcription regulator in the NCBI is probably due to full-length inadequate Psi-BLAST searches biased by the presence of Tetratricopeptide Repeats (TPR) in the large carboxyterminal domain. Figure 5 Predicted PSPPH_2539 protein domain structure based on fold recognition analysis. See text for details on the various structural templates used. Black dots check details connect the C-terminus of one threading domain with the N-terminus of the following domain. Residues 195–300 (green segment) are represented separately as an alternative fold for the N-terminal subdomain of

the full length AAA+ ATPase domain (yellow). The middle part of the protein (Figure 5, yellow area) was found homologous to the AAA+ ATPases (COG3903) based on fold-recognition algorithms and Psi-BLAST searches.

These ATPases are associated with diverse cellular activities and Mannose-binding protein-associated serine protease are able to induce conformational changes in their targets [41]. In the context of the transcription process, AAA+ ATPase domains are involved in the remodeling of σ54 RNA polymerases. Especially the residues 195 to 300 probably possess the receiver or ligand binding domain of the hypothetical transcription factor (green area, Figure 5). TPR-repeats proteins present in P. syringae BLZ945 T3SS-2 Apart from the PSPPH_2539 C-terminal domain, there are two more ORFs, PSPPH_2519 and PSPPH_2523, from the P. syringae pv phaseolicola 1448a T3SS-2 that are predicted to code for proteins that possess TPR domains. TPR domains are typically found in class II chaperones of T3S systems – chaperones of the translocators – as well as in transcriptional regulators of the T3S systems, e.g. the HrpB protein of Ralstonia solanacearum, HilA of Salmonella enterica[42] and SicA, of Salmonella typhimurium involved in the activations of T3SS virulence genes [43]. Proteins with TPR repeats also exist in the Hrc-Hrp2 T3S system of X. campestris (HrpB2 protein) and in the T3S system of Rhizobia (e.g. the 182 residue long Y4yS protein). On the other hand, the Hrc-Hrp1 system of P. syringae does not possess proteins with TPR repeats. DNA characteristics of the P. syringae T3SS-2 gene cluster The T3SS-2 cluster of P.