| Summary: | 碩士 === 中興大學 === 生物化學研究所 === 95 === Xanthomonas campestris pv. campestris is a gram-negative bacterium that is phytopathogenic to cruciferous plants. When infected by Xcc, plants show syndrome of marginal leaf chlorosis, or rotten leafs. During these two years, eight gene targets of predicted functions were chosen for studying. These genes were constructed on vectors with different fusion genes. These targets include XC1894 (flagellar protein FlgD), XC2014 (indigoidine synthesis like protein), XC1015 (HIT like protein), XC3319 (WxcH protein), XC3109 (UDP-galactopyranose mutase), XC930 (transcriptional regulator gntR family), XC1448 (16S rRNA processing protein RimM), and XC6989 (hetI protein). These genes were firstly expressed in His6-tag vector to see if they were soluble. If insoluble, they were further constructed in GST or MBP fusion vector. Through these efforts, XC930 and XC3319 were still insoluble. Further problem was encountered for proteins XC3109, XC1448, and XC6989, which were soluble when fused with MBP, but couldn’t be cleaved successfully using TEV protease. XC2014, XC1894, and XC1015 were the final three soluble proteins with good yield after cleavage, and are our protein targets for further studies.
XC1894 is predicted as a flagellar protein (FlgD) in the Pfam database by BLASTp. It has higher than 40% sequence identity with other flagellar hook capping protein from Stenotrophomonas maltophilia and FlgD from Aeromonas hydrophila. The flagellum can be divided into three portions: 1. The basal body, which is located between the inner and outer membrane and contains eight proteins such as FlgI, FlgJ, FlgF …etc. 2. The hook, which is located outside of outer membrane and consists of FlgE. 3. The filament, which is jointed behind hook region by hook-filament junction protein, FlgK and FlgL, and consists of FliC. The tip of filament is capped by FliD. In previous study, it was found that FlgD can attach to the distal end of rod in basal body and form a cap to prevent FlgE from secreting to extracellular environment directly. FlgD can help FlgE assembly to rapidly extent the length of hook up to 55nm. Subsequently, FlgD slows down its rate of hook elongation by associating with the N terminus of FliK. Finally, FlgD is displaced by FlgK. The replacement of FlgD is the critical step for flagellum to form a mature architecture. The structural determination of FlgD is therefore an important issue to understand how FlgD can determine the hook length. Recently, we have obtained the XC1894 crystal that diffracted to a resolution of 2.5 Å. The structure determination is currently undergoing.
The predicted function of XC2014 is the indigoidine synthase that serves interesting purpose. It was found that plants are infected by Erwinia chrysanthemi, they elicits defense response by generating H2O2 and organic peroxides. The indigoidine is supposed to prevent the damage caused by the generated radical. Although XC2014 has been over-expressed, we still could not find the good crystallization condition. Sequence analysis program such as PONDR revealed a C-terminal disorder region (210-214), which may be responsible for the difficult of crystallization. This region can be deleted to increase the chance of crystallization.
XC1015 was found to belong to the histidine triad (HIT) like protein in the Pfam database. Its homologues were widely found in a variety of organisms, and contain a highly conserved sequence, HψHψHψψ (where ψ is hydrophobic amino acid). Its crystal structure has been determination to a resolution of 1.3 Å. From the determined X-ray structure, XC1015 was found to pose a αββα sandwich fold, and form a homo-dimer through β sheets formation between two monomers. XC1015 structure is similar to the rabbit Hint protein. The most difference between the two structures are their amino acid residues in the catalytic triad, Thr in XC1015 while Ser in rabbit Hint protein.
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