Heterocyst development and diazotrophic metabolism in terminal respiratory oxidase mutants of the cyanobacterium Anabaena sp. strain PCC 7120

Abstract

Heterocyst development was analyzed in mutants of the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 bearing inactivated cox2 and/or cox3 genes, encoding heterocyst-specific terminal respiratory oxidases. At the morphological level, the cox2 cox3 double mutant (strain CSAV141) was impaired in membrane reorganization involving the so-called honeycomb system that in the wild-type strain is largely or exclusively devoted to respiration, accumulated glycogen granules at conspicuously higher levels than the wild type (in both vegetative cells and heterocysts), and showed a delay in carboxysome degradation upon combined nitrogen deprivation. Consistently, chemical analysis confirmed higher accumulation of glycogen in strain CSAV141 than in the wild type. No impairment was observed in the formation of the glycolipid or polysaccharide layers of the heterocyst envelope, consistent with the chemical detection of heterocyst-specific glycolipids, or in the expression of the heterocyst-specific genes nifHDK and fdxH. However, nitrogenase activity under oxic conditions was impaired in strain CSAV135 (cox3) and undetectable in strain CSAV141 (cox2 cox3). These results show that these dedicated oxidases are required for normal development and performance of the heterocysts and indicate a central role of Cox2 and, especially, of Cox3 in the respiratory activity of the heterocysts, decisively contributing to protection of the N(2) fixation machinery against oxygen. However, in contrast to the case for other diazotrophic bacteria, expression of nif genes in Anabaena seems not to be affected by oxygen.

FIG. 1.

Electron micrographs of heterocysts of strains PCC 7120 and CSAV141 (cox2 cox3). Nitrate-grown filaments incubated for 40 h under culture conditions in the absence of combined nitrogen were used for microscopy. HC, “honeycomb” membranes; GG, glycogen granules; GL, glycolipid layer; PL, polysaccharide layer; CB, carboxysomes. Original magnifications, ×12,000 (A and B) and ×30,000 (C).

FIG. 2.

Thin-layer chromatographic separation of lipids extracted from whole filaments (A) or isolated heterocysts (B) of strains PCC 7120 and CSAV141. Cells grown with ammonium (0) or grown with ammonium and incubated in the absence of combined nitrogen for the indicated number of hours, or heterocysts isolated from filaments incubated in the absence of combined nitrogen for the indicated number of hours, were used as described under Materials and Methods. GLI and GLIII, heterocyst envelope glycolipids I and III (10), respectively.

FIG. 3.

Glycogen content of whole filaments (A) or isolated vegetative cells and heterocysts (B) of strains PCC 7120 (wild type [WT]) and CSAV141. Filaments were grown with ammonium and incubated in the absence of combined nitrogen. For panel A, aliquots from each culture were withdrawn at the indicated times for glycogen determination. For panel B, at 48 h vegetative cells and heterocysts were separated as described in Materials and Methods and used for glycogen determination. For each panel, results for representative experiments are presented, from three each that were performed with similar results.

FIG. 4.

Northern blot analysis of the expression of the nifHDK and hesAB-fdxH genes in cox mutant strains. RNA was isolated from cultures of the indicated strains grown with ammonium (0) or grown with ammonium, washed, and incubated in the absence of combined nitrogen for the indicated number of hours. Samples contained 25 to 30 μg of RNA, and hybridizations were carried out with a probe for the nifH or fdxH gene or for the rnpB gene (32), which was used as a loading and transfer control. WT, wild type.