Abstract
Spo0J (ParB), Soj (ParA) proteins and parS DNA sequence were ParAB system implicated in bacterial chromosome segregation. They may work together to accomplish their functions. However, the interaction-relationship and bio-functions between Spo0J and Soj remained largely unclear. In H. pylori, Hp1138 and Hp1139 was putative Spo0J and Soj protein, respectively. However, there were no published data to support their functions. Therefore, it was worth investigating the functions and studying the interactions between these two proteins. To study their functions, recombinant Hp1138 and Hp1139 were constructed, purified and assayed. And high sensitivity and specificity antibodies were prepared. First, the recombinant proteins were characterized and assayed for their individual activity. The data showed that Hp1138 had parS-binding activity and Hp1139 possessed ATPase activity. Together with the possible parS sequences in Hp1142 were found to have binding activity to Hp1138, suggesting ParAB system might exist in H. pylori. Next, three methods were applied to study the interactions between Hp1138, parS and Hp1139. First, ATPase assay indicated that Hp1138 and parS could activate the activity of Hp1139. In addition, the activation by Hp1138 was enhanced in the presence of parS. Second, agarose EMSA showed that Hp1139 could improve parS-binding of Hp1138 and ATP-bound Hp1139 had low parS-binding activity, consistent with that Soj was reported to play a role in the formation of Spo0J-parS complex. Among three states, the ATP-bound Hp1139 was the most efficient effecter. Furthermore, ATP-Hp1139 and Hp1138 could improve parS-binding of each other. The above mentioned agarose EMSA data were new findings in related studies. Third, in Co-IP, the data revealed that Hp1139 could interact with Hp1138 or Hp1138-parS, again the ATP-bound Hp1139 was the most significant interaction partner. However, interaction between Hp1138 and Hp1139 was not found in H. pylori lysate, indicating that a more complicated mechanism might exist in vivo. Finally, protein level and localization of Hp1138 and Hp1139 in H. pylori was determined. Acid-induced analysis showed that these two proteins were not acid-induced protein. Their expression in dividing H. pylori were more than non-dividing H. pylori, suggesting that these two proteins might be involved in cell division. The immuno-fluorescence images displayed that Hp1138 formed discrete foci co-separated with nucleoid and Hp1139 formed helix-like structure through cell division, consistent with previous studies on localization of Spo0J and Soj. The above results suggested that Hp1138 and Hp1139 may be involved in chromosome segregation in H. pylori. However, further experiments were required for studying the bio-functions of Hp1138 and Hp1139 in vivo.