Cloning and expression of a prokaryotic sucrose-phosphate synthase gene from the cyanobacterium Synechocystis sp PCC 6803

Sucrose is one of several low-molecular-weight compounds that cyanobacteria accumulate in response to osmotic stress and which are believed to act as osmoprotectants. The genome of the cyanobacterium Synechocystis sp. PCC 680 3 contains a 2163 bp open reading frame (ORF) that shows similarity to gene s from higher plants encoding sucrose-phosphate synthase (SPS), the enzyme responsible for sucrose synthesis. The deduced amino acid sequence shows 35 -39% identity with known higher-plant SPS sequences. The putative Synechocy stis sps gene was cloned from genomic DNA by PCR amplification and expresse d as a His(6)-tagged amino-terminal fusion protein in Escherichia coli. The expressed protein was purified and shown to be a functional SPS enzyme, co nfirming the identity of the ORF, which is the first sps gene to be cloned from a prokaryotic organism. The Synechocystis SPS has a molecular mass of 81.5 kDa, which is smaller than the typical higher-plant SPS subunit (117-1 19 kDa), and lacks the phosphorylation site motifs associated with light- a nd osmotic stress-induced regulation of SPS in higher plants. The enzyme ha s K-m values for UDPG1c and Fru6P of 2.9 mM and 0.22 mM, respectively, with a V-max of 17 mu mol per minute per mg protein and a pH optimum of 8.5. Un like the higher-plant enzyme, ADPG1c, CDPG1c and GDPG1c can substitute for UDPG1c as the glucosyl donor with K-m values of 2.5, 7.2 and 1.8 mM, respec tively. The enzyme is activated by Mg2+ but not by G1c6P, and is only weakl y inhibited by inorganic phosphate. The purified protein was used to raise a high-titre antiserum, which recognises a low-abundance 81 kDa protein in Synechocystis sp. PCC 6803 extracts. There was no apparent increase in expr ession of the 81 kDa protein when the cells were exposed to moderate salt s tress, and SPS activity was very low in extracts from both unstressed and s alt- stressed cells. These results and the lack of evidence for sucrose acc umulation in Synechocystis sp. PCC6803 lead to the conclusion that expressi on of the sps gene plays no obvious role in adaptation to osmotic stress in this species.