Physiological and molecular studies on the akinete differentiation of filamentous cyanobacteria

Abstract

Akinetes are spore-like cells of filamentous cyanobacteria that allow these organisms to survive long periods of unfavourable conditions. The akinete differentiation and germination processes were studied in detail in two model species of cyanobacterial cell differentiation, the planktonic freshwater Anabaena variabilis ATCC 29413 and the terrestrial or symbiotic Nostoc punctiforme ATCC 29133. The best trigger of akinete differentiation of A. variabilis was low light, while of N. punctiforme was phosphate starvation. Akinetes differed from vegetative cells by their larger size, different cell morphology, and the presence of a large number of intracellular granules. The akinete envelopes of both strains were composed by the same glycolipids as heterocysts and by an exopolysaccharide layer. Also, an unknown and new lipid was detected in lipid extracts of A. variabilis akinetes. During akinete development the storage compounds cyanophycin, glycogen and lipid droplets transiently increased, and the photosynthesis and respiration activities decreased. In both strains, germination was an asynchronous process triggered by light. Fast cell divisions occurred inside of the akinetes envelope and did not need endogenous carbon or nitrogen resources but a functional photosystem to complete the process. Unusual fast heterocyst differentiation was observed during akinete germination in A. variabilis. Fast cell divisions and heterocysts differentiation during germination could have been supported by the high DNA content in akinetes. In the end of the germination process, the akinete envelope was opened (A. variabilis) or partially degraded (N. punctiforme) allowing the emergence of the short filament formed. At this point, the photosynthesis and respiration rates were similar to a vegetative cells culture. Focusing on the specific modulation of the cell wall and cell envelope, proteins were identified that are differentially expressed in akinetes or where shown to be involved in cell differentiation in N. punctiforme. One mutant was created in the ΔAva_2312 gene of A. variabilis encoding a protein with putative function associated to cell wall proteins and characterized. This mutant showed a severe filament dystrophy and it was not able to differentiate heterocysts and to grow without a combined nitrogen source. In addition, a thicker peptidoglycan with more non-crosslinked peptides residues was observed in the septum. Finally, this mutant differentiated akinete-like cells lacking a lipid envelope. Mutants in genes encoding the cell wall lytic enzymes ΔamiC1 and ΔamiC2 of A. variabilis were also created and characterized in this study. Both mutant strains showed filaments of longer vegetative cells, which differentiated non-functional heterocysts and akinete like-cells. The heterocysts with aberrant morphology showed an abnormal or no deposition of glycolipids, absence of cyanophycin polar bodies and accumulation of glycogen characteristic for a phenotype with hampered N2 fixation. Accumulation of a thicker peptidoglycan in the septum between the heterocyst and the vegetative cells was observed in both mutant strains. Moreover, ΔamiC1 and ΔamiC2 mutants formed akinete like-cells with rounded-shape, irregular or missing lipid envelope than in wild type. In this study, clear differences in the metabolic and morphological adaptation mechanisms of akinetes from two important cyanobacterial species were found. For the first time a detailed characterization of the germination process of these species was presented. The results paved the way for further genetic and functional studies of akinete differentiation and germination in these species. In fact, three new mutants in cell wall genes involved in akinete development were characterized showing the relevance of the cell wall proteins in cell differentiation.