Based on the functional characterization of sucrose biosynthesis related proteins [SBP: sucrose-phosphate synthase (SPS), sucrose-phosphate phosphatase (SPP), and sucrose synthase (SuS)] in Anabaena sp. PCC7120 and sequence analysis, we have shown that SBP are restricted to cyanobacterium species and plants, and that they are multidomain proteins with modular architecture. Anabaena SPS, a minimal catalytic SPS unit, defines a glucosyltransferase domain present in all SPSs and SuSs. Similarly, Anabaena SPP defines a phosphohydrolase domain characteristic of all SPPs and some SPSs. Phylogenetic analysis points towards the evolution of modern cyanobacterial and plant SBP from a bidomainal common ancestral SPS-like gene. ß

In higher plants and cyanobacteria, sucrose (Suc) metabolism is carried out by a similar set of enzymes. The function and regulation of Suc metabolism in cyanobacteria has begun to be elucidated. In strains of Anabaena sp., filamentous nitrogen-fixing cyanobacteria, Suc synthase (SuS, EC 2.4.1.13) controls Suc cell level through the cleavage of the disaccharide. The present work shows that there are two sus genes in Anabaena (Nostoc) sp. that are co-regulated regarding the nitrogen source; however, only susA accounts for the extractable SuS activity and for the control of the Suc level. Primer extension analysis has uncovered the sequence of the Anabaena susA and susB ammonium-activated putative promoters, which share a high sequence similarity with that of rbcLS encoding ribulose bisphosphate carboxylase/oxygenase (EC 4.1.1.39) and other ammonium up-regulated genes. Moreover, susA and rbcLS expression is developmentally co-localized to the vegetative cells of the nitrogen-fixing cyanobacterial filaments. Our results strongly suggest the existence of a regulatory network that would coordinate the expression of key genes for Suc and nitrogen metabolism, carbon fixation, and development in Anabaena sp.

Higher plants and cyanobacteria metabolize sucrose (Suc) by a similar set of enzymes. Suc synthase (SuS, UDP-glucose: D: -fructose 2-alpha-D: -glucosyl transferase, EC 2.4.1.13) catalyses the synthesis and cleavage of Suc, and in higher plants, it plays an important role in polysaccharides biosynthesis and carbon allocation. In this work, we have studied the functional relationship between SuS and the metabolism of polysaccharides in filamentous nitrogen-fixing cyanobacteria. We show that the nitrogen and carbon sources and light regulate the expression of the SuS encoding gene (susA), in a similar way that they regulate the accumulation of polysaccharides. Furthermore, glycogen content in an Anabaena sp. mutant strain with an insertion inactivation of susA was lower than in the wild type strain under diazotrophic conditions, while both glycogen and polysaccharides levels were higher in a mutant strain constitutively overexpressing susA. We also show that there are soluble and membrane-bound forms of SuS in Anabaena. Taken together, these results strongly suggest that SuS is involved in the Suc to polysaccharides conversion according to nutritional and environmental signals in filamentous nitrogen-fixing cyanobacteria.

Edited by Miguel De la RosaKeywords: Glucosyltransferase Modular protein Salt treatment Sucrose-phosphate synthase Sucrose-phosphate phosphatase a b s t r a c t It has been reported that higher plants and cyanobacteria synthesize sucrose (Suc) by a similar sequential action of sucrose-phosphate synthase (SPS) and sucrose-phosphate phosphatase (SPP). In the genome of the marine unicellular cyanobacterium Synechococcus sp. PCC 7002 there is a sequence that was not annotated as a putative SPP encoding gene (sppA), although the sequence was available. In this study, we functionally characterize the sppA gene of that strain and demonstrate that it is cotranscribed with spsA, the SPS encoding gene. This is the first report on the coordination of Suc synthesis gene expression in an oxygenic-photosynthetic organism.

The presence of two alkaline/neutral invertases (Inv-A and Inv-B) in the filaments of Nostoc (also named Anabaena) sp. strain PCC 7120 and the involvement of sucrose metabolism in nitrogen fixation led us to investigate the physiological function of those isoforms in cells growing under different nitrogen sources. The highest expression level of each encoding gene was obtained in the presence of ammonium. These results were paralleled by polypeptide and enzyme activity level. In cells of N(2)-fixing filaments, localization of gene expression and subcellular enzyme activity assays demonstrated that invA gene (alr1521) expresses only in vegetative cells, whereas for invB (alr0819), expression is detected in both vegetative cells and heterocysts. In contrast to invA, when invB was knocked out, the filaments were unable to grow on diazotrophic conditions and the accumulation of sucrose and glycogen was altered. Our results demonstrate an essential role for Inv-B for diazotrophic growth and that Inv-B plays a key role in the coordination of sucrose and glycogen metabolism. We can also suggest that invB is likely to integrate the repertoire of genes regulated by a cyanobacterial transcription factor (NtcA) that plays a central role in global nitrogen control.