| Abstract |
Type III secretion systems (T3SSs) are essential mediators of the interaction of many Gram-negative bacteria with plant or animal eukaryotic cells. The T3SS pathway of plant pathogens is encoded by hrp (HR and pathogenicity) and hrc (HR and conserved) genes. The Hrc proteins direct secretion of T3SS substrates across the bacterial envelope, whereas a subset of the Hrp proteins, whose function is only partially defined, are themselves secreted by the T3SS and direct the translocation of effectors through host cell barriers. HrpG is a hypothetical class I T3SS chaperone as predicted by fold recognition algorithms and Pseudomonas syringae T3SS genes organization. Small angle X-ray scattering and circular dichroism experiments were used for evaluation of this hypothesis. In contradistinction to the homodimeric class I T3SS chaperones, HrpG forms a tetramer in solution. However the protein particle does not possess the symmetry of a closed tetrameric complex. Deletion of the 14 carboxyterminal residues led to the formation of a stable dimer in solution with the appropriate symmetry. Although the dimeric particle possesses the overall shape of class I T3SS chaperones, we could not conclusively propose that HrpG belongs to this class of chaperones because of its longer diameter and the higher content of α-helix. Moreover, HrpG was found to interact with HrpO and the carboxyterminal domain of HrcU through native electrophoresis. HrpO is a highly soluble protein of the T3SS of P. syringae pv phaseolicola. Size exclusion chromatography profiles resemble those obtained by FliJ (Fraser et al. 2003). Thus, an aberrant elution profile was also demonstrated for HrpO in the present work. HrpO was found to have an extended shape in solution by SAXS with low melting temperature by CD experiments. During the thermal unfolding experiments no intermediate population of the protein is observed. The high content of secondary structure however, discourages the classification of HrpO with intrinsically unfolded proteins (IUp). Class I T3SS chaperones bind to the N-terminal part of effectors and deliver them to the T3SS ATPase for translocation. The avrPphF operon of P. syringae pv phaseolicola possesses two open reading frames, a class I T3SS chaperone (ORF1) and its cognate virulence effector (ORF2). Although each of the proteins crystallizes quite easily, the complex was recalcitrant to crystallization. Here we present the preliminary crystallographic analysis of the AvrPphF ORF1 crystals as well as the solution structure of the AvrPphF ORF1/ORF2 complex based on small angle X-ray scattering data.
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Δομικά χαρακτηριστικά υποψηφίων σαπερονών του εκκριτικού συστήματος τύπου III κα των συμπλόκων τους
