Background

Filamentous cyanobacteria, such as Anabaena variabilis ATCC 29413 and Nostoc punctiforme ATCC 29133, are widely used as model organisms for the study of multicellularity and their capability to differentiate heterocysts and akinetes. Under favorable conditions, these photosynthetic Gram-negative bacteria grow in the form of filaments, which are composed of hundreds of vegetative cells. In response to insufficient supply of organically combined nitrogen, about 10% of semi-randomly spaced cells can differentiate into nitrogen-fixing heterocysts, which provide the filaments with nitrogen (Figure 1A) (Fay, 1992; Muro-Pastor and Maldener, 2019). Akinetes are spore-like resting cells that differentiate from the vegetative cells in response to diverse environmental conditions, including changes in light intensity and quality, temperature, and nutrient deficiency (Figure 1B).

Figure 1. Light micrographs showing cell differentiation in A. variabilis. (A) Vegetative filament containing heterocyst, indicated by black arrowhead. (B) Akinetes induced under low light condition. Scale bars, 10 µm.

Both cell types—heterocysts and akinetes—are characterized by the presence of a thick multilayered envelope, mainly composed of exopolysaccharides and glycolipids (HGLs) (Maldener et al., 2014; Perez et al., 2016; Garg and Maldener, 2021b). The glycolipids, which form laminated layers in the heterocyst and akinete envelopes, are of great interest due to their different functions in these two cell types. In heterocysts, they act as an oxygen diffusion barrier to protect the oxygen labile enzyme nitrogenase from oxygen (Fay, 1992; Wolk et al., 1994), while in akinetes, glycolipids protect the cells from various stress factors, such as freezing, desiccation, and lysozyme attack (Garg and Maldener, 2021a).

Mutants with aberrantly formed heterocyst envelopes are unable to grow without alternative sources of combined nitrogen. Many of those lose the ability to synthesize HGLs or to transport them beyond the cell wall to the heterocyst envelope (Ernst et al., 1992; Fiedler et al., 1998; Fan et al., 2005). The akinete envelope shares some structural similarities with the heterocyst envelope, suggesting that akinetes are the evolutionary ancestors of heterocysts (Wolk et al., 1994; Perez et al., 2018). To better understand this evolutionary relationship, a prerequisite is the elucidation of the composition of these special cells’ envelopes. Additionally, mutants showing phenotypes related to survival under environmental stress or starvation could be checked for the correct laminated layer composition using the protocol presented here.

Here, we use the thin-layer chromatography (TLC) protocol that was previously established by Nichols and Wood in 1968; with this method, they identified glycolipids, which are specific for the heterocyst forming cyanobacteria (Nichols and Wood, 1968). Later, the Wolk group showed that these glycolipids constitute the laminated layer in the envelope of heterocysts (Winkenbach et al., 1972). The two major heterocyst specific glycolipids in Anabaena species are characterized as 1-α-glucosyl-3,25-hexacosanediol (HG₂₆-diol) and its 3-ketotautomer (HG₂₆-keto-ol). Their separation via TLC is dependent on their polarity. We utilized this method with a few modifications for the analysis of the akinete specific glycolipids (Perez et al., 2018; Garg and Maldener, 2021a). We present a reproducible and reliable TLC protocol to analyze the glycolipids present in the heterocyst and akinete envelopes. This protocol includes the easy extraction of lipids using regular solvents, such as methanol and chloroform, and their clear separation and visualization on the TLC plate, which can be scanned directly. Furthermore, this method allows the extraction of specific glycolipids from the TLC plate in a manner that subsequent analysis of their chemical composition by HPLC-MS analysis is possible, as previously described (Perez et al., 2018; Shvarev et al., 2018).