Catabolic Function of Compartmentalized Alanine Dehydrogenase in the Heterocyst-Forming Cyanobacterium Anabaena sp. Strain PCC 7120

In the diazotrophic filaments of heterocyst-forming cyanobacteria, an exchange of metabolites takes place between vegetative cells and heterocysts that results in a net transfer of reduced carbon to the heterocysts and of fixed nitrogen to the vegetative cells. Open reading frame alr2355 of the genome of Anabaena sp. strain PCC 7120 is the ald gene encoding alanine dehydrogenase. A strain carrying a green fluorescent protein (GFP) fusion to the N terminus of Ald (Ald-N-GFP) showed that the ald gene is expressed in differentiating and mature heterocysts. Inactivation of ald resulted in a lack of alanine dehydrogenase activity, a substantially decreased nitrogenase activity, and a 50% reduction in the rate of diazotrophic growth. Whereas production of alanine was not affected in the ald mutant, in vivo labeling with [¹⁴C]alanine (in whole filaments and isolated heterocysts) or [¹⁴C]pyruvate (in whole filaments) showed that alanine catabolism was hampered. Thus, alanine catabolism in the heterocysts is needed for normal diazotrophic growth. Our results extend the significance of a previous work that suggested that alanine is transported from vegetative cells into heterocysts in the diazotrophic Anabaena filament.Cyanobacteria such as those of the genera Anabaena and Nostoc grow as filaments of cells (trichomes) that, when incubated in the absence of a source of combined nitrogen, present two cell types: vegetative cells that perform oxygenic photosynthesis and heterocysts that perform N₂ fixation. Heterocysts carry the oxygen-labile enzyme nitrogenase, and, thus, compartmentalization is the way these organisms separate the incompatible activities of N₂ fixation and O₂-evolving photosynthesis (9). In Anabaena and Nostoc, heterocysts are spaced along the filament so that approximately 1 in 10 to 15 cells is a heterocyst. Heterocysts differentiate from vegetative cells in a process that involves execution of a specific program of gene expression (12, 15, 39). In the N₂-fixing filament, the heterocysts provide the vegetative cells with fixed nitrogen, and the vegetative cells provide the heterocysts with photosynthate (38). Two important aspects of the diazotrophic physiology of heterocyst-forming cyanobacteria that are still under investigation include the actual metabolites that are transferred intercellularly and the mechanism(s) of transfer (10).Because the ammonium produced by nitrogenase is incorporated into glutamate to produce glutamine in the heterocyst and because the heterocyst lacks the main glutamate-synthesizing enzyme, glutamine(amide):2-oxoglutarate amino transferase (GOGAT; also known as glutamate synthase), a physiological exchange of glutamine and glutamate resulting in a net transfer of nitrogen from the heterocysts to the vegetative cells has been suggested (21, 36, 37). On the other hand, a sugar is supposed to be transferred from vegetative cells to heterocysts. Because high invertase activity levels are found in the heterocysts (34) and because overexpression of sucrose-degrading sucrose synthase in Anabaena sp. impairs diazotrophic growth (4), it is possible that sucrose is a transferred carbon source. Indeed, determination of ¹⁴C-labeled metabolites in heterocysts isolated from filaments incubated for short periods of time with [¹⁴C]bicarbonate identified sugars and glutamate as possible compounds transferred from vegetative cells to heterocysts (13). However, this study also identified alanine as a metabolite possibly transported from vegetative cells to heterocysts.The cyanobacteria bear a Gram-negative type of cell envelope, carrying an outer membrane (OM) outside the cytoplasmic membrane (CM) and the peptidoglycan layer (9, 15). In filamentous cyanobacteria, whereas the CM and peptidoglycan layer surround each cell, the OM is continuous along the filament, defining a continuous periplasmic space (10, 19). In Anabaena sp. strain PCC 7120, the OM is a permeability barrier for metabolites such as glutamate and sucrose (27). Two possible pathways for intercellular molecular exchange in heterocyst-forming cyanobacteria have been discussed: the periplasm (10, 19) and cell-to-cell-joining proteinaceous structures (11, 22, 25). Whereas the latter would mediate direct transfer of metabolites between the cytoplasm of adjacent cells, the former would require specific CM permeases to mediate metabolite transfer between the periplasm and the cytoplasm of each cell type (10).In Anabaena sp. strain PCC 7120, two ABC-type amino acid transporters have been identified that are specifically required for diazotrophic growth (29, 30). The N-I transporter (NatABCDE), which shows preference for neutral hydrophobic amino acids, is present exclusively in vegetative cells (30). The N-II transporter (NatFGH-BgtA), which shows preference for acidic and neutral polar amino acids, is present in both vegetative cells and heterocysts (29). A general phenotype of mutants of neutral amino acid transporters in cyanobacteria is release into the culture medium of some hydrophobic amino acids, especially alanine (16, 23, 24), which is accumulated at higher levels in the extracellular medium of cultures incubated in the absence than in the presence of a source of combined nitrogen (30).Thus, alanine is a conspicuous metabolite in the diazotrophic physiology of heterocyst-forming cyanobacteria, and the possibility that it moves in either direction between heterocysts and vegetative cells has been discussed (13, 29, 30). Alanine dehydrogenase, which catalyzes the reversible reductive amination of pyruvate, has been detected in several cyanobacteria (8). In Anabaena spp., alanine dehydrogenase has been found at higher levels or exclusively in diazotrophic cultures (26), and in the diazotrophic filaments of Anabaena cylindrica it is present at higher levels in heterocysts than in vegetative cells (33). Open reading frame (ORF) alr2355 of the Anabaena sp. strain PCC 7120 genome is predicted to encode an alanine dehydrogenase (14). In this work we addressed the expression and inactivation of alr2355, identifying it as the Anabaena ald gene and defining an important catabolic role for alanine dehydrogenase in diazotrophy.