Synechocystis sp. PCC6803, a unicellular cyanobacterium, can utilize light energy to grow autotrophically or glucose to grow heterotrophically, and can be naturally transformed by exogenous DNA. It is one of the model species for the research of photosynthesis in cyanobacteria. In this study, photoautotrophic mutants were obtained by screening from random insertion mutants of Synechocystis sp. PCC6803, some genes that are potentially involved in photoautotrophy were identified, and the genes ssr2060 and slr2094 were further investigated for their roles in photoautotrophic growth.
The main researches and results are in three parts:
(1) Selection of mutants of photoautotrophic growth and identification of the mutated genes. Two random insertion mutant libraries of about 11,000 clones were constructed and screened, from which 28 mutants were selected that were incapable of growing photoautotrophically. By using inverse PCR, 13 genes were identified by sequencing the flanking DNA and searching the genome sequence of Synechocystis sp. PCC6803. Among these genes, ssr2060 is predicated to encode a function-unknown protein and the rest 12 genes encode known proteins.
(2) The study of gene ssr2060 whose function is unknown. The reconstruction, targeted disruption of downstream ssr2061 and slr1238 and complementation experiments all showed that gene ssr2060 is essential for photoautotrophic growth of Synechocystis sp. PCC6803. Ssr2060 is predicated to be a transmembrane protein. The comparison of Ssr2060 with other cyanobacterial homologs showed that their structures are similar. Chl a fluorescence at room temperature suggested that the photosynthetic electron transport is defective in the mutant. By using the different donors and acceptors to check the flow of electrons through the photosynthetic electron transport, the inactivation of ssr2060 might block the electron flow from oxygen-evolving complex to QA. Fluorescence emission spectroscopy at 77K indicated that the mutation of gene ssr2060 is not involved in the assembly of PS II complex, while the conversion to state 1 is impaired. The mutant’s photoautotrophic growth at different light intensities, O2 evolution at different light wavelength and light intensities proved that energy transfer is not affected by the mutation of gene ssr2060.
(3) The study of gene slr2094. The gene slr2094 encodes fructose-1, 6-/sedoheptulose-1,7-bisphosphatase (fbpI) in Synechocystis sp. PCC6803. The mutant was reconstructed and showed that gene slr2094 is indispensable for photoautotrophic growth. No enayme activity of fructose-1,6-bisphosphatase was detected in the mutant. The mixotrophic growth of mutant and the gene expression by using reporter gene luxAB at different light intensities showed that the mutant could grow mixtrophically only under low light intensity.
The main conclusions of this study are:
(1) ssr2060 (encoding a transmembrane protein) is an essential gene for photoautotrophic growth of Synechocystis sp. PCC6803. In the mutant, the electron transport from oxygen-evolving complex to QA is blocked and the conversion to state 1 is inhibited, while the mutation of ssr2060 doesn’t affect the assembly of PS II and the energy transfer.
(2) slr2094 (encoding fructose-1, 6-/sedoheptulose-1,7-bisphosphatase (fbpI)) is essential to photoautotrophic growth of Synechocystis sp. PCC6803, and the mutant could not grow mixtrophically under high light intensity.