Identification of novel regulatory mechanisms controlling heterocyst development in Anabaena Sp. strain PCC 7120

The regulatory mechanisms that govern heterocyst development in Anabaena sp. strain PCC 7120 have been continuously refined over the last two decades. In this work, we show that three of the sigma factor genes present in the Anabaena sp. strain PCC 7120 genome are developmentally regulated. Time-lapse microscopy of gfp reporter strains indicated that expression of sigC, sigG, and sigE is upregulated specifically in differentiating cells at 4 h, 9 h and 16 h, respectively, after induction of heterocyst development. We proposed that the sigma factors encoded by these genes are involved in regulation of heterocyst-specific genes whose expression is relatively coincident with that of sigC, sigG, or sigE. Indeed, inactivation of the sigC gene caused delayed and reduced expression of genes required for the early stages of heterocyst development, and caused delayed development. Inactivation of the sigE gene caused a considerable drop in expression of nifH, a late gene required for nitrogen fixation. We also provide evidence that c-di-GMP, a novel bacterial second messenger, is involved in regulating heterocyst development. The all2874 gene encodes a bona fide diguanylate cyclase, which synthesizes c-di-GMP, and the gene's inactivation resulted in a decreased tendency to form heterocysts; this phenotype was exacerbated by high light intensity. We hypothesize that the putative operon all2875-all2874 senses and relays information about light conditions and this information is integrated into the decision to form heterocysts. Finally, we identified the all0187 gene, which is expressed at 9 h, a time when cells that have initiated differentiation commit to complete the process. In nitrogen-free medium, all0187 mutant filaments formed abnormally long heterocysts and were unable to grow diazotrophically. Septum formation between heterocysts and their flanking vegetative cells was incomplete, leaving one or both poles of the heterocysts more opened and potentially more permeable to oxygen. Despite having nitrogenase activity, the all0187 mutant was unable to grow diazotrophically. We hypothesize that the diazotrophic growth defect is caused by the inability of the heterocysts to transport fixed nitrogen to the neighboring vegetative cells.