Xiaoling Miao, Qingyu WU*

1. Sucrose accumulation in salt-stressed cells of agp gene deletion-mutant in cyanobacterium Synechocystis sp. PCC 6803 Xiaoling Miao, Qingyu WU* Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, P.R.China Fig. 2. Influence of 0.9 M NaCl shock on photosynthesis (▲), specific growth rate (●) and growth (○) of the wild type (A,B,C) and the agp- mutant cells (D,E,F). Cell growth was determined by monitoring the optical density of the culture at 730 nm. The specific growth rate was defined as an increase of OD730 during the time indicated. The rate of photosynthesis was monitored as oxygen evolution in intact cells. Upon addition of NaCl at a concentration of 0.9 M, net O2 evolution and growth rate of the wild type cells declined during the first hours (A,B), with greatest depression after about 10 h, therefore there was only a little increase in OD730 within the first 10 h (C). Thereafter a gradual recovery of growth and photosynthesis led to salt-acclimated cells. For the agp- mutant cells, the changes in growth and O2 evolution were almost similar to that of the wild type cells after the salt shock (D,E,F), but it took about 48 h, not 10 h, for the mutant to recover its growth and photosynthesis. Fig. 1. Mutation of the agp gene homolog in Synechocystis sp. PCC 6803. The PCR-amplified Synechocystis 6803 DNA fragment was digested with BamHI and SacI, and cloned in pUC118. The resulting plasmid was named pUCA (A). A 0.3 kb KpnI-SmaI fragment within this Synechocystis 6803 agp gene was deleted and replaced by an erythromycin resistance cartridge (Erm) from plasmid pRL425. The resulting plasmid was designated as pUCAE (B). The pUCAE plasmid was used to transform the Synechocystis 6803 wild type strain. The mutant was designated as agp-. (C): Physical map indication of DNA fragments between WT and agp-. Fig. 4.Contents of osmoprotective substances glucosylglycerol and sucrose in the agp- mutant and wild type (WT) cells after cultivation in the medium with 0 M (Controls), 0.9 M and 1.0 M NaCl for 48 h respectively. No glucosylglycerol was detected in WT and in agp- as control and in agp- after 0.9 M and 1.0 M NaCl shocking. As compared to 0.9 M salt shock, the content of glucosylglycerol increased 34% after 1.0 M salt shock for 48 h. However, the content of sucrose in the mutant cells only increased 14% after 1.0 M NaCl shock for 48 h. The total amount of sucrose (50 µg ml-1 OD730-1) reached only 75% of the glucosylglycerol content (67 µg ml-1 OD730-1) of the wild type cells after 1.0 M NaCl shock for 48 h, not as 88% after 0.9 M NaCl shock. This is probably the main reason responsible for the mutant unable to tolerate 1.0 M salt concentration. Fig. 3.Influence of 1.0 M NaCl shock on oxygen evolution (A) and growth (B) of the wild type (▲) and the agp- mutant cells (●). Unlike 0.9 M salt shock, 1.0 M salt shock resulted in a lag phase of growth between 10 and 24 h in the wild type cells. The O2 evolution and the growth of the wild type cells recovered gradually after 24 h, while the O2 evolution of the mutant decreased to 0 after 48 h and the growth stopped at the same time. These results showed that the mutant could tolerate up to about 0.9 M salt concentration. FEMS Microbiology Letters 218:71-77, 2003 Abstract Synechocystis sp. PCC 6803 is a moderately halotolerant cyanobacterium. It accumulates glucosylglycerol after a salt shock and tolerates up to 1.2 M NaCl. The agp gene encoding the ADP-glucose pyrophosphorylase is involved in cyanobacterial glycogen synthesis and glucosylglycerol formation. By in vitro DNA recombination technology, a mutant with partial deletion of agp gene in the cyanobacterium Synechocystis sp. PCC 6803 was constructed This mutant could not synthesize glycogen or the osmoprotective substance glucosylglycerol. Upon the block-up of the glycogen biosynthesis pathway, the carbon flow may towards the direction of UDP-glucose formation, which would then used in the reaction of sucrose-phosphate-synthase to make sucrose. The mutant could also tolerate high salt concentration if sucrose might act as a potent osmoprotectant as glucosylglycerol. To test this hypothesis, the strain of agp gene deletion-mutant of Synechocystis sp. PCC 6803 was grown in the presence of 0.9 M NaCl in the medium. After 48 h of incubation, no glucosylglycerol was detected and the total amount of sucrose was 26 times of that of in wild type cells. The total amount of sucrose reached 88% of the glucosylglycerol content of the wild-type cells after 48 h. Therefore the agp- mutant could tolerate up to 0.9 M salt concentration.Our results suggest that sucrose might act as a similar potent osmoprotectant as glucosylglycerol in cyanobacterium Synechocystis sp. PCC 6803. There has been currently considerable interest in the growth and mass culture of cyanobacteria as a biomass source. An understanding of the mechanisms of salinity tolerance in cyanobacteria and their effects on the chemical composition of cells to be used as a source of biomass is therefore of particular value in determining the suitability of natural waters for the mass culturing of them. Thus, information on the organic solubles produced by cyanobacteria as osmotica may facilitate the choice of suitable cyanobacterial strains for mass culturing or other biotechnological uses in specific ecosystems. *Corresponding author.Tel: +86 (10) 62781825); Fax: +86 (10) 62781825. E-mail: qingyu@tsinghua.edu.cn