In the cyanobacterium Synechocystis 6803, many genes of unknown function are essential for basic life activities. Insertion inactivation of these genes could lead to loss of viability, and the mutants could not be completely segregated, consequently, genetic studies are hard to perform with these genes. However, to construct mutants expressing these genes from a copper-regulated promoter P-petE may result in complete segregation. To find out the physiological effects of the investigated gene, copper may be removed from the medium to turn off the gene expression or adjusted to certain low concentrations to lower the gene expression level. This report presents the construction of the P-petE-sll0260 mutant and the study on the essential role of sll0260 in Synechocystis 6803. Upstream of the P-petE promoter was an omega cassette with stem-loop structures at both ends to terminate background transcriptions. The mutant was completely segregated in the presence of cupric ion as shown with PCR detection. In the mutant, the expression of sll0260 was fully turned on in the presence of 320nmol/L cupric ion, but turned off after removal of cupric ion from the medium. The copper regulation of sll0260 by P-petE was confirmed by Western blot analysis. Upon copper deprivation, the growth of the P-petE-sll0260 strain was seriously inhibited, pigments were greatly reduced, thylakoid membranes were destructed, oxygenic photosynthesis ceased and respiration was remarkably lowered. Under the same conditions, the wild type strain showed almost no change in growth and physiological characters. All these results suggested that sll0260 should be essential for the maintenance of basic life activities of Synechocystis 6803. Based on Western blot analysis, sll0260-encoded protein was localized to the total membrane fraction. Further analysis with purified thylakoid membranes and a mixture of cytoplasmic and outer membranes showed that the sll0260-encoded membrane protein (Sll0260) was located in either cytoplasmic or outer membrane or both, but not in the thylakoid membranes. The predicted amino acid sequence of Sll0260 showed a low level similarity to certain ion transporters. We proposed that Sll0260 could be involved in ion transport or the biogenesis of thylakoid membranes.
Luo Quan (firstname.lastname@example.org) ; Xu Xu-Dong (email@example.com).THE ESSENTIAL ROLE OF ORF SLL0260 IN MAINTENANCE OF BASIC LIFE ACTIVITIES OF SYNECHOCYSTIS SP PCC6803,Acta Hydrobiologica Sinica,2009,33(5):789-794