hetC, a gene coding for a protein similar to bacterial ABC protein exporters, is involved in early regulation of heterocyst differentiation in Anabaena sp. strain PCC 7120.

Transposon-generated mutant C3 of Anabaena sp. strain PCC 7120 is unable to form heterocysts upon deprivation of combined nitrogen but forms a pattern of spaced, weakly fluorescent cells after 2 days of deprivation. Sequence analysis of chromosomal DNA adjacent to the ends of transposon Tn5-1058 in mutant C3 showed a 1,044-amino-acid open reading frame, designated hetC, whose predicted protein product throughout its C-terminal two-thirds has extensive similarity to the HlyB family of bacterial protein exporters. Its N-terminal third is unique and does not resemble any known protein. hetC lies 1,165 bp 5' from the previously described gene hetP. Reconstruction of the C3 mutation and its complementation in trans with a wild-type copy of hetC confirmed that hetC has an essential regulatory role early in heterocyst development. hetC is induced ca. 4 h after nitrogen stepdown, hours after induction of hetR. Expression of hetC depends on HetR and may depend on HetC. Highly similar sequences are present 5' from the initiation codons and in the 3' untranslated regions of hetC and of two heterocyst-specific genes, devA and hetP.     

Overexpression of pknE blocks heterocyst development in Anabaena sp. strain PCC 7120

The upstream intergenic regions for each of four genes encoding Ser/Thr kinases, all2334, pknE (alr3732), all4668, and all4838, were fused to a gfpmut2 reporter gene to determine their expression during heterocyst development in the cyanobacterium Anabaena (Nostoc) sp. strain PCC 7120. P(pknE)-gfp was upregulated after nitrogen step-down and showed strong expression in differentiating cells. Developmental regulation of pknE required a 118-bp upstream region and was abolished in a hetR mutant. A pknE mutant strain had shorter filaments with slightly higher heterocyst frequency than did the wild type. Overexpression of pknE from its native promoter inhibited heterocyst development in the wild type and in four mutant backgrounds that overproduce heterocysts. Overexpression of pknE from the copper-inducible petE promoter did not completely inhibit heterocyst development but caused a 24-h delay in heterocyst differentiation and cell bleaching 4 to 5 days after nitrogen step-down. Strains overexpressing pknE and containing P(hetR)-gfp or P(patS)-gfp reporters failed to show developmental regulation of the reporters and had undetectable levels of HetR protein. Genetic epistasis experiments suggest that overexpression of pknE blocks HetR activity or downstream regulation.     

Effect of controlled expression of the hetR gene on heterocyst formation in the filamentous cyanobacterium Anabaena sp. PCC 7120

hetR is a central regulatory gene inducing and possibly maintaining irreversible heterocyst differentiation in filamentous cyanobacteria. A plasmid was constructed which enabled IPTG-mediated, controlled expression of hetR from a p _tac promoter in Anabaena . When introduced into a heterocyst-deficient hetR mutant, induction led to massive formation of heterocysts in a medium free of combined nitrogen. In nitrate-containing cultures, induction elicited formation of only a few heterocysts, but led to nitrogen chlorosis in vegetative cells as evidenced from degradation of phycobiliproteins. Removal of the inducer IPTG caused chlorosis and death of the organisms in nitrate-free medium, but no reversal of heterocyst formation. This indicates that constant synthesis of HetR is not the (sole) reason for irreversibility of heterocyst formation.