Identification Of The OriC Region And Its Influence On Heterocyst Development In The Filamentous Cyanobacterium Anabaena Sp. Strain PCC 7120

DNA replication is one of the most important events in the cell cycle. The replication initiation protein DnaA and DNA replication origin oriC are main constituents in DNA replication initiation. The DNA replication in E. coli has been studied thoroughly, but very little work has been published about cyanobacteria.In this study, the oriC region of Anabaena sp. strain PCC 7120 was predicted by bioinformatic method. The oriC region is located between dnaA and dnaN in the chromosome, and this 415-bp region contains six DnaA boxes and an AT-rich region. This region was ligated to pRL271Ω, a non-replicative vector in cyanobacteria, to generate pRL271ΩoriC. Then it was transferred to Synechocystis PCC 6803 by conjugation. After total DNA was extracted from the exconjuguants, several methods have been used to prove that the original plasmid containing oriC region of Anabaena PCC 7120 can replicate in Synechocystis PCC 6803 autonomously. Firstly, several primers were designed to do overlapping PCR, two round of overlapping PCR was done. Secondly, the total DNA was transformed into E. coli, then plasmid DNA was extracted and digested to confirm the plasmid. Thirdly, we transferred the total DNA into wild type Synechocystis PCC 6803, and extracted total DNA from the exconjuguants, then PCR was done to confirm that the original plasmid was in the exconjuguants. The fourth method was Southern blotting. The last method was ligation of the oriC region to another vector-pBluescript, and then it was transferred into Synechocystis PCC 6803. Total DNA was extracted from exconjuguants, and transformed to E.coli, then plasmid DNA was extracted and digested to confirm the plasmid. In summary, we confirmed that the oriC region from Anabaena PCC 7120 predicted by bioinformatic method is a functional origin to initiate DNA replication.Anabaena sp. strain PCC 7120 is a filamentous, nitrogen-fixing cyanobacterium. Upon deprivation of combined nitrogen, about 5-10% of the cells can differentiate to heterocysts to perform nitrogen fixation. There is little work had been published about the relationship between DNA replication initiation and heterocyst differentiation. In this study, we ligated the oriC region and the whole dnaA-oriC-dnaN region to the vector pRL271Ωrespectively, to genarate vector pRL271ΩoriC and pRL271ΩdnaAN, and then transferred them to Anabaena PCC 7120 by conjugation. The exconjuguants were named MoriC and MdnaAN. Their total DNA was extracted and PCR results indicated that the oriC region integrated with the chromosome of Anabaena PCC 7120 through homologous recombination, so there are two copies of oriC region and dnaA-oriC-dnaN region in the chromosome of MoriC and MdnaAN.After cultured in nitrogen free medium, the heterocyst differentiation of MoriC and MdnaAN was abnormal compared with the wild type. The rate of growth was slowed down, there are normal heterocysts 24 hours after combined nitrogen step-down, but mutiple continuous pro-heterocysts could be seen after prolonged incubation (after 48 hours).Real-time RT-PCR showed that expression of two heterocyst-related genes, hetR and hetN, was altered in these strains comparing with the wild type. These results suggest that the balance between oriC and DnaA could be important for heterocyst development. In our study, we found that if the replication initiation region on the vector integrated into the chromosome, abnormal phenotypes of heterocysts development can be observed.