Heterocyst-forming cyanobacteria are multicellular organisms in which growth requires the activity of two metabolically interdependent cell types, the vegetative cells that perform oxygenic photosynthesis and the dinitrogen-fixing heterocysts. Vegetative cells provide the heterocysts with reduced carbon, and heterocysts provide the vegetative cells with fixed nitrogen. Heterocysts conspicuously accumulate polar granules made of cyanophycin [multi-L-arginyl-poly (L-aspartic acid)], which is synthesized by cyanophycin synthetase and degraded by the concerted action of cyanophycinase (that releases β-aspartyl-arginine) and isoaspartyl dipeptidase (that produces aspartate and arginine). Cyanophycin synthetase and cyanophycinase are present at high levels in the heterocysts. Here we created a deletion mutant of gene all3922 encoding isoaspartyl dipeptidase in the model heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120. The mutant accumulated cyanophycin and β-aspartyl-arginine, and was impaired specifically in diazotrophic growth. Analysis of an Anabaena strain bearing an All3922-GFP (green fluorescent protein) fusion and determination of the enzyme activity in specific cell types showed that isoaspartyl dipeptidase is present at significantly lower levels in heterocysts than in vegetative cells. Consistently, isolated heterocysts released substantial amounts of β-aspartyl-arginine. These observations imply that β-aspartyl-arginine produced from cyanophycin in the heterocysts is transferred intercellularly to be hydrolyzed, producing aspartate and arginine in the vegetative cells. Our results showing compartmentalized metabolism of cyanophycin identify the nitrogen-rich molecule β-aspartyl-arginine as a nitrogen vehicle in the unique multicellular system represented by the heterocyst-forming cyanobacteria. intercellular communication | nitrogen fixation

Cyanobacteria are oxygenic phoautotrophs that can utilize inorganic nitrogen salts, atmospheric nitrogen and some amino acids such as arginine as nitrogen source. Under unbalanced growth in the presence of sufficient nitrogen, many cyanobacteria accumulate cyanophycin, a co-polymer of aspartate and arginine that serves as a nitrogen reservoir. Cyanophycin metabolism enzymes include cyanophycin synthetases, cyanophycinase and isoaspartyl dipeptidase, which splits aspartyl arginine released from cyanophycin by cyanophycinase into aspartate and arginine. The arginine catabolic pathway of cyanobacteria has been recently elucidated and consists of two bifunctional enzymes, arginine-guanidine removing enzyme (AgrE) and proline oxidase (PutA). This pathway makes available to metabolism the four nitrogen atoms of arginine, three as ammonia and one as glutamate. A variant of the pathway cycles ornithine (an intermediate in the AgrE-catalyzed reactions) back to arginine incorporating aspartate and, hence, recovering its nitrogen atom for metabolism. Many cyanobacteria also make use of this pathway to utilize arginine taken up from the outer medium through a high-affinity ABC transporter. An analysis of co-occurrence in cyanobacteria of the genes encoding enzymes of cyanophycin metabolism, arginine catabolism and the arginine and aspartate transporters indicates a strong correlation between the presence of cyanophycin and the AgrE/PutA pathway.