Functional Analysis For An Unknown Encoding Gene Of Synechocystis Sp. Strain PCC 6803 And Its Optimation For Natural Transformation Conditions

Cyanobacteria are autotrophic prokaryotes performing oxygenic photosynthesis similar to that of higher plants. Synechocystis sp. strain PCC 6803 (Synechocystis 6803) is a unicellular cyanobacterium. Although Synechocysits 6803 complete genome is first characterized in phothosynthetic organisms, up to now, there exist many encoding genes of unknown functions in this unicellular cyanobacterium, including some potential unknown genes for regulating the photosystem II complex. However, little is known regarding the characteristics of these unknown genes in Synechocystis 6803.The results of yeast two-hybrid indicated that there exist a protein-protein interaction between the expression product of an unknown function (rps2) and the subunit Psb27 of photosystem II. It has been previously reported that the subunit Psb27 is a key factor for assembling and stabilizing the oxygen-evolving complex of photosystem II. To reveal whether the product of rps2 influenced the assembly and stability of oxygen-evolving complex of photosystem II, therefore, the investigation of rps2 gene was conducted in this study. First of all, the homologous recombination vector, pUC-Δrps2, was constructed, and then this vector was transferred into wild type Synechocystis 6803 by using the natural transformation method. After several subcultures, the transformations were examined by using the PCR. The experimental results indicated that the rps2 gene inactition mutant was obtained. Secondly, the photochemical and nonphotochemical quenching and oxygen-evolving activity of photosystem II between the rps2 gene inactivation mutant (Δrps2) and wild-type Synechocystis 6803 (WT) were determined by using Dual-PAM-100 and Clark-type oxygen electrode, respectively. The results indicated that the rps2 gene inactiviation in Syenchocystis 6803 significantly affected the activity of oxygen-evolving of photosystem II. Thirdly, the growth curves ofΔrps2 and WT were measured by using photometer. The results indicated that the rps2 gene inactiviation in Syenchocystis 6803 significantly affected the rate of cell growth. Taken together, these results suggested that the activity of oxygen-evolving complex of photosystem II was down-regulated by the rps2 gene via changing the expression level of Psb27 subunit, thereby inhibiting the rate of cell growth.During the natural transformation for the homologous recombination vector, pUC-Δrps2, we accidently found that treatment with low light significantly enhances the frequency of natural transformation whereas high light illumination remarkably shortens the appearance time of Synechocystis 6803 transformants. Moreover, the transformation efficiency of Synechocystis 6803 was closely associated with the permeability of the cell membranes. Further, the molecular mechanism for natural transformation of Synechocystis 6803 was discussed.In conclusion, the activity of oxygen-evolving complex of photosystem II was down-regulated by the unknown rps2 gene, thereby inhibiting the rate of cell growth. Therefore, the expression product of rps2 gene is another key factor for stabilizing the oxygen-evolving complex of photosystem II. In addition, the intensity of light is an important factor for influencing the efficiency of natural transformation in Synechocystis 6803.