Analysis Of Pattern Formation In The Heterocyst Forming Filamentous Cyanobacterium Anabaena Sp Strain Pcc 7120 PDF Download

The planktonic freshwater filamentous cyanobacterium Anabaena variabilis ATCC 29413 (syn. Trichormus variabilis) grows as filaments of hundreds of cells and is capable of differentiating nitrogen fixing heterocysts, motile hormogonia and dormant akinetes from vegetative cells in response to different stress conditions. Under conditions of nitrogen limitation, heterocysts form in a semi-regular pattern and provide the filaments with organic nitrogen by fixing N2. Akinetes are transient spore-like cells enabling these bacteria to withstand harsh environmental conditions. When suitable growth conditions are available, the akinetes can germinate and produce new vegetative filaments, thereby providing cyanobacteria with a means of survival in changing habitats. Heterocysts and akinetes are characterized by the presence of a thick multilayered envelope, including an outermost polysaccharide and an inner glycolipid layer. Until now, the role of a glycolipid layer, which reduces the entry of oxygen into the heterocysts for the maintenance of a microoxic environment and nitrogen fixation, was unknown in spore-like akinetes. Therefore, in this work, the function of the gene Ava_2595 in A. variabilis, which is homolog to the known hglB gene, that encodes a putative polyketide synthase involved in heterocyst glycolipid synthesis in Anabaena sp. PCC 7120, a species which does not form akinetes, was elucidated. The hglB mutant was created and its phenotype was characterized and further investigated for the functionality of heterocysts and akinetes. This work revealed that the hglB mutant strain formed aberrant heterocysts and akinete-like cells, in which the specific glycolipid layers were absent demonstrating the requirement of HglB in glycolipid layer formation in both heterocyst and akinete envelope. Consequently, the mutant was unable to fix N2 under aerobic condition and to grow diazotrophically. This study also confirmed that both cell types use a glycolipid of identical chemical composition in their special envelopes. Furthermore, we unraveled the role of the glycolipids in protecting the akinetes against harsh conditions, like freezing, desiccation, oxidative stress and lytic enzymes. Severely reduced tolerance to stress conditions was exhibited by the akinetes lacking the glycolipids but under standard conditions, they could germinate normally. Our study established the dual role of the glycolipid layer in fulfilling different functions in the evolutionary-related specialized cells of cyanobacteria and indicated the existence of a common biosynthetic pathway for glycolipid synthesis in heterocysts and akinetes involving the same gene hglB. Akinetes accumulate large quantities of cytoplasmic reserve products, mainly glycogen and the nitrogen storage polymer cyanophycin during their differentiation. In this work, the physiological function of cyanophycin in akinete differentiation and germination was investigated which showed that the cyanophycin production is not required for these cellular processes. This study also summarized the significant morphological and physiological changes that occur during formation and germination of the akinetes in A. variabilis ATCC 29413. Further analysis of changes occurring during akinete formation and germination using scanning electron microscopy (SEM) found that the mature akinetes have a wrinkled envelope and during germination, the surface of the envelope smoothens upon increase in the cell size, and finally, the akinete envelope ruptures to release the short emerging filament. Also, in this work, the akinete envelope architecture of different layers, the exopolysaccharide and glycolipid layer, could be visualized and showed that this multilayered envelope helps to withstand the osmotic stress and to maintain the structural integrity. Finally, we demonstrated that the intercellular communication decreased during akinete formation as compared to the vegetative cells. In contrast, the cell communication was restored in freshly germinating filaments.