Sucrose metabolism in Microcystis aeruginosa PCC 7806

M.A KOLMAN; C.M. BERÓN; L.E. GIARROCCO; G.L. SALERNO

Montreal, Quebec, Canada

Simposio; 13th International Symposium on Phototrophic Prokaryotes; 2009

ISPP - International Society of Phototrophic Prokaryotes

<!-- /* Font Definitions */ @font-face {font-family:Calibri; mso-font-alt:"Century Gothic"; mso-font-charset:0; mso-generic-font-family:swiss; mso-font-pitch:variable; mso-font-signature:-1610611985 1073750139 0 0 159 0;} /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin-top:0cm; margin-right:0cm; margin-bottom:10.0pt; margin-left:0cm; line-height:115%; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman"; mso-fareast-font-family:Calibri; mso-ansi-language:EN-US; mso-fareast-language:EN-US;} @page Section1 {size:612.0pt 792.0pt; margin:70.85pt 3.0cm 70.85pt 3.0cm; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> Introduction. Sucrose is one of the most common non-reducing disaccharides in Nature. It has been extensively studied in plants, where it is the main transport photosynthetic product, a source of carbon and energy, and a molecule associated with environmental stress and signal transduction. During the last decade, Suc metabolism was demonstrated in cyanobacteria. Suc is synthesized through a two-step pathway involving SPS (U/ADP-glucose:D-fructose-6-phosphate 2-a-D-glucosyltransferase) and SPP (sucrose-6F-phosphate-phospho-hydrolase), either in unicellular or filamentous cyanobacterium strains. However, Suc cleavage by SuS (A/UDP-glucose:D-fructose 2-a-D-glucosyl transferase), has only been reported in filamentous heterocyst-forming cyanobacteria, where Suc was demonstrated to play a crucial role in N2 fixation and in polysaccharide production. Objective. The aim of this study was to investigate the role of Suc in Mycrocystis aeruginosa, a unicellular non-N2 fixing strain, well known as one of the most common bloom-forming cyanobacteria in fresh water environments. Methods. M. aeruginosa PCC 7806 was cultured in BG11 medium. DNA fragments corresponding to Suc metabolism candidate genes were PCR amplified. The open reading frames (orfs) were ligated into the pRSET vector. The recombinant His–tagged proteins were expressed in Escherichia coli BL21 (DE3)pLysS. Proteins were purified by Ni2+ affinity chromatography, while protein extracts and purification, enzyme activities and expression analyses were performed as reported previously for Anabaena [Cumino et al. 2007; Curatti et al. 2008]. Results. We searched in the M. aeruginosa genome for sequences homologous to those of functionally characterized genes coding for SPS, SPP and SuS. We retrieved three contiguous orfs in the contig 328: IPF_1566, IPF_1564, and IPF_1565 whose deduced amino-acid sequences are about 55%, 53%, and 72% identical to the protein sequences of Anabaena sp. PCC 7120 SPP SPS, and SuS-A, respectively. The resulting His-tagged fusion proteins showed SPP, SPS, and SuS activity. In addition, cell free extracts from M. aeruginosa were chormatographed through an ion exchange column and the enzyme activities were assayed in the eluted fractions. When we studied the biochemical properties of the enzymes, we found that SPS specificity was similar to that of other cyanobacterial SPSs, and interestingly, SuS activity could only be measured in the Suc cleavage direction. Expression analyses by RT-PCR and Northern blotting indicated that the three genes are transcribed during standard culture conditions of the cyanobacterium. Conclusions. This is the first report showing that both Suc synthesis and cleavage take place in a potential toxic cyanobacterium. Remarkably, we showed that SuS is also present in a unicellular, non-N2-fixing strain. The fact that this enzyme acts only in the Suc cleavage direction suggests that it might correspond to a novel class of SuS. Further studies on this enzyme may shed some light into its reaction mechanism and into the role of Suc in Microcystis cells.   Cumino AC, Marcozzi C, Barreiro R, Salerno GL. Plant Physiol. 143, 1385-1397 (2007) Curatti L, Giarrocco LE, Cumino AC, Salerno GL. Planta 228, 617-625 (2008)