| dc.description.abstract |
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. |
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