Compartmentalized cyanophycin metabolism in the diazotrophic filaments of a heterocyst- forming cyanobacterium Mireia Burnat, Antonia Herrero, and Enrique Flores1 Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas and Universidad de Sevilla, E-41092 Seville, Spain Edited by Robert Haselkorn, University of Chicago, Chicago, IL, and approved January 24, 2014 (received for review October 2, 2013) Heterocyst-forming cyanobacteria are multicellular organisms in cyanobacterial filament (4, 6). Indeed, when incubated under which growth requires the activity of two metabolically interde- proper conditions, isolated heterocysts can produce glutamine pendent cell types, the vegetative cells that perform oxygenic that is released to the surrounding medium (6). In addition to photosynthesis and the dinitrogen-fixing heterocysts. Vegetative glutamate, two compounds that can be transferred from vege- cells provide the heterocysts with reduced carbon, and heterocysts tative cells to heterocysts are alanine and sucrose (8). Alanine provide the vegetative cells with fixed nitrogen. Heterocysts con- can be an immediate source of reducing power in the heterocyst, spicuously accumulate polar granules made of cyanophycin [multi- where it is metabolized by catabolic alanine dehydrogenase, the L-arginyl-poly (L-aspartic acid)], which is synthesized by cyano- product of ald (9). Sucrose, a universal vehicle of reduced carbon phycin synthetase and degraded by the concerted action of in plants, appears to have a similar role within the diazotrophic cyanophycinase (that releases β-aspartyl-arginine) and isoaspartyl cyanobacterial filament, because diazotrophic growth requires dipeptidase (that produces aspartate and arginine). Cyanophycin that sucrose is produced in vegetative cells and hydrolyzed by a synthetase and cyanophycinase are present at high levels in the specific invertase, InvB, in the heterocysts (10–13). Sucrose is a heterocysts. Here we created a deletion mutant of gene all3922 more important carbon vehicle to heterocysts than alanine, be- encoding isoaspartyl dipeptidase in the model heterocyst-forming cause inactivation of invB (12, 13) has a stronger negative effect cyanobacterium Anabaena sp. strain PCC 7120. The mutant accu- on diazotrophic growth than inactivation of ald (9). In summary, mulated cyanophycin and β-aspartyl-arginine, and was impaired there is evidence for transfer of sucrose, glutamate, and alanine specifically in diazotrophic growth. Analysis of an Anabaena strain from vegetative cells to heterocysts and of glutamine in the reverse bearing an All3922-GFP (green fluorescent protein) fusion and de- direction. termination of the enzyme activity in specific cell types showed that Heterocysts bear inclusions in the form of refractile granules isoaspartyl dipeptidase is present at significantly lower levels in that are located at the cells poles (close to the heterocyst heterocysts than in vegetative cells. Consistently, isolated hetero- “necks”) and are made of cyanophycin [multi-L-arginyl-poly (L- cysts released substantial amounts of β-aspartyl-arginine. These aspartic acid)], a nitrogen-rich polymer (14, 15). Although pro- observations imply that β-aspartyl-arginine produced from cyano- duction of cyanophycin is not required for diazotrophic growth phycin in the heterocysts is transferred intercellularly to be hydro- (16, 17), its conspicuous presence in the heterocysts suggests lyzed, producing aspartate and arginine in the vegetative cells. Our a possible role of this cell inclusion in diazotrophy (18, 19), for results showing compartmentalized metabolism of cyanophycin instance as a nitrogen buffer to balance nitrogen accumulation identify the nitrogen-rich molecule β-aspartyl-arginine as a nitro- and transfer (20). Cyanophycin is synthesized by cyanophycin gen vehicle in the unique multicellular system represented by the synthetase that adds both aspartate to the aspartate backbone of heterocyst-forming cyanobacteria. the polymer and arginine to the β-carboxyl group of aspartate residues in the backbone (21, 22). Cyanophycin is degraded by intercellular communication | nitrogen fixation MICROBIOLOGY Significance eterocyst-forming cyanobacteria represent a unique group Hof multicellular bacteria in which growth requires the ac- The heterocyst-forming cyanobacteria represent a true multi- tivity of two interdependent cell types: the vegetative cells that cellular system found in the bacterial world that adds unique perform oxygenic photosynthesis and the N2-fixing heterocysts features to biodiversity. They grow as chains of cells containing (1, 2). Vegetative cells fix CO2 photosynthetically and transfer two metabolically interdependent cellular types, the vegeta- reduced carbon to heterocysts (3), in which nitrogenase fixes N2 tive cells that fix carbon dioxide performing oxygenic photo- producing ammonia that is incorporated into amino acids via the synthesis and the dinitrogen-fixing heterocysts. Heterocysts glutamine synthetase−glutamate synthase (GS/GOGAT) path- accumulate a cell inclusion, cyanophycin [multi-L-arginyl-poly way (4). Heterocysts transfer fixed N to the vegetative cells in the (L-aspartic acid)], as a nitrogen reservoir. We have found that filament (5). In a cyanobacterium such as the model organism the second enzyme of cyanophycin catabolism, isoaspartyl di- Anabaena sp. strain PCC 7120 (hereafter Anabaena), the fila- peptidase, is present at significantly higher levels in vegetative ment can be hundreds of cells long. Under nitrogen fixing con- cells than in heterocysts, identifying the nitrogen-rich dipeptide ditions, which are established when no combined nitrogen is β-aspartyl-arginine as a nitrogen vehicle to the vegetative cells available in the medium (1, 2), about one in 10–20 cells are het- and showing that compartmentalization of a catabolic route is erocysts, which are semiregularly distributed along the filament used to improve the physiology of nitrogen fixation. ensuring an even exchange of nutrients between the two cell types. A full understanding of this multicellular system needs the iden- Author contributions: M.B., A.H., and E.F. designed research; M.B. performed research; M.B., tity of the intercellularly exchanged compounds to be known. A.H., and E.F. analyzed data; and E.F. wrote the paper. Because the heterocysts bear high levels of glutamine synthe- The authors declare no conflict of interest. tase but lack glutamate synthase (6, 7), an exchange of glutamine This article is a PNAS Direct Submission. (transferred from heterocysts to vegetative cells) for glutamate 1To whom correspondence should be addressed. E-mail: [email protected]. (transferred from vegetative cells to heterocysts) has been pro- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. posed for the GS/GOGAT cycle to work in the diazotrophic 1073/pnas.1318564111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1318564111 PNAS | March 11, 2014 | vol. 111 | no. 10 | 3823–3828 Downloaded by guest on September 30, 2021 cyanophycinase that releases a dipeptide, β-aspartyl-arginine (23), the mutant chromosomes (Fig. S1). Strain CSMI6, which was which is hydrolyzed to aspartate and arginine by an isoaspartyl selected for further analysis, exhibited weak growth under diaz- dipeptidase homologous to plant-type asparaginases (24). Cya- otrophic conditions on solid medium, although it grew well in the nophycin synthetase is the product of cphA (21) and cyanophy- presence of combined nitrogen, either nitrate or ammonium cinase of cphB (23). Anabaena bears two gene clusters, cph1 and (shown in Fig. 1B for nitrate-supplemented medium). Comple- cph2, each containing both cphA and cphB genes, of which cph1 mentation of CSMI6 with a plasmid bearing wild-type all3922 contributes most to cyanophycin metabolism (17). All these genes (see SI Materials and Methods) allowed diazotrophic growth are expressed in vegetative cells and heterocysts, but differential corroborating that growth impairment resulted from the all3922 expression leads to levels of both cyanophycin synthetase and mutation (see strain CSMI6-C in Fig. 1B). Determination of cyanophycinase that are much higher (about 30- and 90-fold, growth rate constants in liquid culture showed that the growth respectively) in heterocysts than in vegetative cells (25). The fate rate of CSMI6 in the presence of combined nitrogen (nitrate or of the β-aspartyl-arginine released in the cyanophycinase reaction ammonium) was comparable to that of the wild type, but it was has, however, not been investigated until now. about 46% slower in the absence of combined nitrogen (Table 1). As deduced from heterologous expression in Escherichia coli, Nitrogenase activity, measured by the acetylene reduction assay, ORF all3922 of the Anabaena chromosome encodes an isoaspartyl was only 15% lower in the mutant than in the wild type (Table 1). dipeptidase (24). In this work, we have generated Anabaena mu- Microscopic observation of strain CSMI6 showed the presence tants of all3922, including inactivation and reporter-expressing of abundant granulation in the cytoplasm of the cells (Fig. 1C). strains, and have found that all3922 is required for optimal diazo- To test the possibility that the granulation corresponded to cya- trophic growth and that isoaspartyl dipeptidase is present mainly in nophycin, cyanophycin granule polypeptide (CGP) isolation was the vegetative cells of the diazotrophic filament. Our