Relationship Between The Heterocyst Development And DNA Replication In Anabaena Sp. Strain PCC 7120

Protein intein is widespread in a variety of organisms. Several intein elements are also present in cyanobacteria, and some of them have been studied biochemically in vitro. However, no evidence is available for intein removal in vivo in cyanobacteria. In the filamentous cyanobacterium Anabaena sp. strain PCC 7120, the DNA replication factor DnaE is encoded by two split open reading frames (ORFs) far apart from each other on the chromosome and each of them could contain a split intein element. This organism can undergo a developmental process leading to the formation of nitrogen-fixing cells, heterocysts. Heterocysts are terminally differentiated cells with arrest of cell cycle. Since DnaE is an important cell cycle element involved in DNA replication, we would like to provide in vivo evidence for DnaE intein removal in cyanobacteria and determine whether mature DnaE protein is still present in heterocyst. In this study, we showed that the products of these two ORFs were joined together to form a complete DnaE protein through the process of protein trans-splicing. More interestingly, protein trans-splicing could be detected in vivo for the first time in cyanobacteria, which allowed us to compare the formation of mature DnaE protein in heterocysts and vegetative cells and showed that mature DnaE protein could be formed in both cell types. Transcriptional fusion between the promoter regions of the two split ORFs and gfp reporter also demonstrate that both ORFs are transcribed in vegetative cells and heterocysts, without strong variation during the process of heterocyst differentiation. Although heterocysts are terminally differentiated and may not replicate its chromosome, the expression and maturation of DnaE in these cells may underlie the need of DNA replication machinery in processes such as DNA recombination and repair.DnaA is the initiator protein in DNA replication, and is essential for cell cycle. We are interested in studying its expression in both cell types. The result showed that the expression of DnaA was fluctuant during the development of heterocyst, which may suggest that DnaA undertake other functions or were also expressed during the proheterocysts. The temperature-sensitive strains have been screened out in Escherichia coli and Streptomyces, which were helpful to obtain synchronous culture. We attempted to screen temperature-sensitive Anabaena PCC 7120 strain through site-directed mutagenesis in dnaA in order to study the relationships between DNA replication and heterocyst development. The first screening results showed that Anabaena PCC 7120 strains after single crossover are sensitive at 39℃.