Identification of the active site of HetR protease and its requirement forheterocyst differentiation in the cyanobacterium Anabaena sp strain PCC 7120

HetR is a serine-type protease required for heterocyst differentiation in h eterocystous cyanobacteria under conditions of nitrogen deprivation. We hav e identified the active Ser residue of HetR from Anabaena sp. strain PCC 71 20 by site-specific mutagenesis.By changing the S152 residue to an Ala resi due, the mutant protein cannot be labeled by Dansyl fluoride, a specific se rine-type protein inhibitor. The mutant protein showed no autodegradation i n vitro. The mutant hetR gene was introduced into Anabaena strain 884a, a h etR mutant. The resultant strain, Anabaena strain S152A, could not form het erocysts under conditions of nitrogen deprivation even though the up-regula tion of the mutant hetR gene was induced upon removal of combined nitrogen. The Anabaena strain 216, which carries a mutant hetR gene encoding S179N H etR and could not form heterocysts, also produced HetR protein upon inducti on. Sequence comparison shows that Ser152 is conserved in all cyanobacteria l HetR. Immunoblotting was used to study HetR induction in both the wild-ty pe and mutant strains. The amount of mutant HetR in strain S152A and in str ain 216 increased continuously for 24 h after nitrogen step-down, while the amount of HetR in wild-type cells reached a maximum level within 6 h after nitrogen step-down. Our results show the Ser152 is the active site of HetR . The protease activity is required for heterocyst differentiation and migh t be needed for repression of HetR overproduction under conditions of nitro gen deprivation.