The Regulatory Mechanism Of The Alr1550 Gene During Heterocyst Differentiation In Anabaena SP.PCC 7120

Anabaena sp.PCC 7120 is a kind of multicellular filamentous cyanobacteria which can perform biological nitrogen fixation.Anabaena respond to the external cue of nitrogen scarcity by the differentiation of heterocysts,cells specialized in the fixation of atmospheric nitrogen.The differentiation of heterocyst is an extremely complex physiological process,which involves multiple external and internal signals.Among those most important regulators for heterocyst development,NtcA and HetR function as master stimulators,while PatS and HetN function as master inhibitors.They regulated the expression of other heterocyst development related genes directly or indirectly,so that heterocysts follow a spatial pattern along the filament.A recent study in our laboratory showed that the expression of alr1550 was up-regulated in response to nitrogen deprivation and restricted predominantly in heterocysts.In vivo and in vitro experiment confirmed that the expression of alr1550 gene is regulated directly by NtcA,Inactivation of alr1550 significantly increased heterocyst frequency after nitrogen deprivation.These results verified elementarily that alr1550 gene may be a new heterocyst differentiation regulation factor.In order to explore how alr1550 regulates the differentiation of heterocyst in the cell,we mainly focused on the following aspects.We further detected the expression level of Alr1550 in the absence of combined nitrogen by Western-blotting.The results showed that the level of Alrl550 was significantly increased and the up regulation of Alrl550 occured solely in heterocysts.Next,the expression levels of hetR,patS and hetN were detected in the alr1550 disruption mutants.Compared with the wild type,the transcription level of hetR in the mutant was up-regulated(about 1.5 folds);the hetN transcription and translation levels were significantly down regulated,while the patS expression level was slightly up-regulated.These facts suggested that the inactivation of Air 1550 affected the expression of key genes of heterocyst differentiation.The interaction between Air 1550 and HetR was confirmed by Far-western blotting and Bacterial two hybrid experiments.In the mobility shift assay,we found that Alk1550 protein can specifically promote HetR binding to PhetP.Competition experiment results showed that Alr1550 protein can compete with PatS-6 for the interaction with HetR,and thus remove the inhibition of PatS on HetR function.Overall,it is concluded that Alr1550 may be a novel regulatory factor,which may interact with the HetR protein in the cell and regulate the heterocyst differentiation.