FraH is required for reorganization of intracellular membranes during heterocyst differentiation in Anabaena sp. strain PCC 7120

Abstract

In the filamentous, heterocyst-forming cyanobacteria, two different cell types, the CO(2)-fixing vegetative cells and the N(2)-fixing heterocysts, exchange nutrients and regulators for diazotrophic growth. In the model organism Anabaena sp. strain PCC 7120, inactivation of fraH produces filament fragmentation under conditions of combined nitrogen deprivation, releasing numerous isolated heterocysts. Transmission electron microscopy of samples prepared by either high-pressure cryo-fixation or chemical fixation showed that the heterocysts of a ΔfraH mutant lack the intracellular membrane system structured close to the heterocyst poles, known as the honeycomb, that is characteristic of wild-type heterocysts. Using a green fluorescent protein translational fusion to the carboxyl terminus of FraH (FraH-C-GFP), confocal microscopy showed spots of fluorescence located at the periphery of the vegetative cells in filaments grown in the presence of nitrate. After incubation in the absence of combined nitrogen, localization of FraH-C-GFP changed substantially, and the GFP fluorescence was conspicuously located at the cell poles in the heterocysts. Fluorescence microscopy and deconvolution of images showed that GFP fluorescence originated mainly from the region next to the cyanophycin plug present at the heterocyst poles. Intercellular transfer of the fluorescent tracers calcein (622 Da) and 5-carboxyfluorescein (374 Da) was either not impaired or only partially impaired in the ΔfraH mutant, suggesting that FraH is not important for intercellular molecular exchange. Location of FraH close to the honeycomb membrane structure and lack of such structure in the ΔfraH mutant suggest a role of FraH in reorganization of intracellular membranes, which may involve generation of new membranes, during heterocyst differentiation.

Fig. 1.

Ultrastructure of heterocysts in Anabaena sp. strain PCC 7120 (a and c) and the ΔfraH mutant, strain CSVT4 (b and d). Filaments grown in BG11 medium and incubated for 48 h in BG11₀ medium (in the absence of combined nitrogen) were prepared for electron microscopy by high-pressure cryo-fixation (a and b) or chemical fixation (c and d) as described in Materials and Methods. Hep, heterocyst envelope polysaccharide; Hgl, heterocyst glycolipid layer; CP, cyanophycin plug; HC, honeycomb intracellular membrane structure. (The cyanophycin plug is frequently lost, leaving a white, empty space in the micrographs, during sample preparation after chemical fixation.) Scale bar, 1 μm.

Fig. 2.

Ultrastructure of aberrant cells in a ΔfraH mutant. Filaments of strain CSVT4 (ΔfraH) grown in BG11 medium and incubated for 48 h in BG11₀ medium (in the absence of combined nitrogen) were prepared for electron microscopy by high-pressure cryo-fixation as described in Materials and Methods. A transmission electron micrograph of one sample showing cell doublets (gray arrows) and a dividing cell (white arrow) within a thickened cell envelope is presented; an isolated heterocyst can also be observed (black arrow). CP, cyanophycin plug.

Fig. 3.

Localization of FraH-C-GFP in the Anabaena filaments. (A) Sequence features of FraH. The region with Cys-X-X-Cys motifs is shown in red, the Pro-rich region is shown in blue, and one possible transmembrane segment is shown in gray (topology according to MEMSAT3 Prediction [http://bioinf.cs.ucl.ac.uk/psipred/]). The position of a putative FHA domain is also indicated. The GFP is not to scale. (B, C, and D) Filaments of Anabaena sp. strain CSVT26 from bubbled cultures grown with nitrate (BG11C; NO₃⁻) or incubated in the absence of combined nitrogen for 24 h (BG11₀C; N₂) were visualized by confocal (B and D) or fluorescence (C) microscopy as described in Materials and Methods. The micrographs show overlays of the cyanobacterial red autofluorescence and the GFP fluorescence. Arrows in panel B point to cells in which the helical pattern of FraH-C-GFP is evident. Arrows in panel D point to an intercalary heterocyst (1), a terminal heterocyst (2), and a proheterocyst presumably released from a broken filament (3). Scale bar, 3 μm.

Fig. 4.

Localization of FraH-C-GFP during development of heterocyst-containing filaments. Filaments of Anabaena sp. strain CSVT26 grown with nitrate were incubated in the absence of combined nitrogen for 24 h and visualized by confocal microscopy at the indicated times as described in Materials and Methods. Bright field (left), GFP fluorescence (center), and overlays of red cyanobacterial autofluorescence and GFP fluorescence (right) are shown. Identical microscope settings were used for the different time points.

Fig. 5.

Localization of FraH-C-GFP in heterocysts. Filaments of Anabaena sp. strain CSVT26 grown with nitrate were incubated in the absence of combined nitrogen for 24 h and analyzed by fluorescence microscopy and deconvolution of images. Portions of filaments with a terminal (A) or an intercalary (B) heterocyst are shown. Top, GFP fluorescence; middle, red cyanobacterial autofluorescence and GFP fluorescence overlay; bottom, bright field and GFP fluorescence overlay.

Fig. 6.

Intercellular transfer of 5-carboxyfluorescein in Anabaena sp. strain PCC 7120 and the ΔfraH mutant. (A) Examples of 5-carboxyfluorescein transfer between vegetative cells in nitrate-grown filaments of strains PCC 7120 (wild type) and CSVT4 (ΔfraH). The arrows indicate the places of bleaching. Fluorescence was then checked at about 2-s intervals showing different degrees of recovery. (B) Recovery rate constant, R, was determined for vegetative cells in nitrate-grown filaments or filaments incubated without combined nitrogen for 24 h (dinitrogen). Values are the means and standard deviation of the means (n, number of filaments in which FRAP was quantified). Scale bar, 3 μm.

Fig. 7.

Intercellular transfer of calcein between vegetative cells and heterocysts of Anabaena sp. strain PCC 7120 and the ΔfraH mutant. (A) Examples of calcein transfer between vegetative cells and heterocysts of strains PCC 7120 (wild type) and CSVT4 (ΔfraH). Nitrate-grown filaments incubated for 24 h in the absence of combined nitrogen. The lower panel shows an isolated heterocyst subjected to partial bleaching. The arrows indicate the places of bleaching. (B) Exchange coefficient, E, was determined for calcein transfer from vegetative cells to heterocysts in filaments incubated without combined nitrogen for 24 h or 48 h. Values are the means and standard deviation of the means (n, number of filaments in which FRAP was quantified). Scale bar, 3 μm.