PrpJ1 And PrpJ2, Two Phosphatases In Anabaena Sp. PCC 7120: Their Roles In Oxidative Adaption And Substrates/Partners In Vivo

The filamentous cyanobacterium Anabaena sp. strain PCC 7120 is an important model organism for the research of prokaryotic cell differentiation. When the growth medium is deprived of combined nitrogen source,5-10% of the cells along each filament are induced to differentiate into heterocysts, which are specialized in nitrogen fixation and supply nitrogen source to other cells. There are two homologous genes, prpJl and prpJ2, in Anabaena sp.strain PCC7120, both of which encode PP2C-type protein phosphatases.The prpJl mutant, S20, grows poorly in BG11. When deprived of a combined nitrogen source, S20 can differentiate into heterocysts, but is not able to grow. The heterocysts of S20 lack one of the two heterocyst-specific glycolipids, and are severely detached from filaments. Although the deletion of prpJ2 has no significant effect on the heterocysts differentiation, the heterocyst development is completely inhibited in the double mutant of prpJl and prpJ2. prpJl is involved in the biosynthesis of one of the two heterocyst-specific glycolipids, and prpJ2 may play a role at the early stage of heterocysts differentiation.We found that prpJ1 and prpJ2 were involved in the response of Anabaena sp.strain PCC7120 to oxidative stress. The oxidant MV and H2O2 were used to test the sensitivity of WT, S19, S20 and S19/S20. Compared to WT, S20 and S19 exhibited slightly more resistant to MV, while S19/S20 was much more resistant to MV. Both prpJl and prpJ2 were detected to be down regulated in WT under oxidative conditions using qPCR. The above data indicate that prpJ1 and prpJ2 are implicated in oxidative response regulation in Anabaena sp.strain PCC 7120.Two methods, Pull down and 2-D electrophoresis, have been employed to identify the substrates or interacting proteins of PrpJl and PrpJ2 in this study. Both PrpJl and PrpJ2 are membrane protein, which are difficult to be purified. Therefore, we only expressed the soluble parts (a His-tag was added at the N-terminal or at the C-terminal) of the two proteins in E. coli. Using the purified proteins as the baits, we performed Pull down assays using the whole cellular proteins isolated from either WT or mutant cells. Unfortunately, we could not identify the proteins that interact with PrpJl or PrpJ2 in this way. However, we noticed that PrpJl could be site-specifically cleaved when incubated with the cell lysate of S20 cells that were harvested after 24 h diazotrophic growth. With the 2-D electrophoresis experiments, we further checked the proteins phosphorylated in either WT or the mutants. Our result showed that the mutation of prpJl and/or prpJ2 did affect the phosphorylation levels of some proteins, which could be the substrates of PrpJl or PrpJ2 in vivo. Next, the differentially phosphorylated proteins are to be identified by mass spectrum.