Septum-localized protein required for filament integrity and diazotrophy in the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120

Abstract

Heterocysts, formed when filamentous cyanobacteria, such as Anabaena sp. strain PCC 7120, are grown in the absence of combined nitrogen, are cells that are specialized in fixing atmospheric nitrogen (N(2)) under oxic conditions and that transfer fixed nitrogen to the vegetative cells of the filament. Anabaena sp. mutants whose sepJ gene (open reading frame alr2338 of the Anabaena sp. genome) was affected showed filament fragmentation and arrested heterocyst differentiation at an early stage. In a sepJ insertional mutant, a layer similar to a heterocyst polysaccharide layer was formed, but the heterocyst-specific glycolipids were not synthesized. The sepJ mutant did not exhibit nitrogenase activity even when assayed under anoxic conditions. In contrast to proheterocysts produced in the wild type, those produced in the sepJ mutant still divided. SepJ is a multidomain protein whose N-terminal region is predicted to be periplasmic and whose C-terminal domain resembles an export permease. Using a green fluorescent protein translationally fused to the carboxyl terminus of SepJ, we observed that in mature heterocysts and vegetative cells, the protein is localized at the intercellular septa, and when cell division starts, it is localized in a ring whose position is similar to that of a Z ring. SepJ is a novel composite protein needed for filament integrity, proper heterocyst development, and diazotrophic growth.

FIG. 1.

Scheme showing the different domains of SepJ and the approximate location of the fusion to GFP. The solid bar denotes the region of the protein that corresponds to the DNA fragment cloned for the generation of mutant SR2787a.

FIG. 2.

Filament lengths of Anabaena sp. and SR2787a. Filaments grown in BG11 medium (in the presence of 5 μg Em/ml for the mutant) were harvested, washed with nitrogen-free medium, and resuspended in BG11 or BG11₀ medium with antibiotic for the mutant. After incubation for the indicated times at 30°C in the light on a shaker, samples, which were taken with great care to prevent disruption, were visualized by standard light microscopy. Ninety to 120 filaments were counted in each case. WT, wild type.

FIG. 3.

Ultrastructures of strain SR2787a incubated in nitrogen-free medium for 48 h (a, b) and of a heterocyst of wild-type Anabaena sp. induced for 48 h in nitrogen-free medium (c). Size markers, 1 μm. C, carboxysomes; Hep, heterocyst polysaccharide layer; Hgl, heterocyst glycolipid layer; Sep, septum; S. c., small cell.

FIG. 4.

Lack of heterocyst glycolipids in mutant strain SR2787a. Filaments of Anabaena sp. (wild type [WT]) and SR2787a incubated in BG11₀ medium for 48 h were harvested, and lipids were extracted and analyzed by thin-layer chromatography. Arrowheads indicate heterocyst-specific glycolipids.

FIG. 5.

Expression of sepJ. (a) RNA was isolated from bubbled cultures of Anabaena sp. grown with ammonium, nitrate, or N₂ as the nitrogen source and hybridized with a probe containing nucleotides −35 to +1475 of sepJ with respect to the ATG gene start codon. (b) RNA was isolated from bubbled cultures of wild-type Anabaena sp. (WT) and its derivatives CSE2 (ntcA) and 216 (hetR). Strains were either grown with ammonium (time zero) or grown with ammonium and incubated in the absence of combined nitrogen for the indicated numbers of hours. RNA was hybridized with a probe containing nucleotides +505 through +1475 of sepJ with respect to the ATG gene start codon. In both cases, hybridization with a probe of the rnpB gene was used as a loading and transfer control (lower panels). Similar results were obtained when the hybridization shown in panel a was performed with the shorter probe.

FIG. 6.

Subcellular localization of SepJ-GFP. (a) Light transmission (left panels), cyanobacterial autofluorescence (middle panels), and GFP fluorescence (right panels) micrographs of ammonium-grown, nitrate-grown, and N₂-grown filaments of strain CSAM137. Size marker, 6 μm. Heterocysts do not autofluoresce. (b, c) Light transmission (top panels), cyanobacterial autofluorescence (middle panels), and GFP fluorescence (bottom panels) micrographs of cells of N₂-grown (b) or nitrate-grown (c) filaments of strain CSAM137. Size markers, 3 μm.