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

HetR is a serine-type protease required for heterocyst differentiation in heterocystous cyanobacteria under conditions of nitrogen deprivation. We have identified the active Ser residue of HetR from Anabaena sp. strain PCC 7120 by site-specific mutagenesis. By changing the S152 residue to an Ala residue, the mutant protein cannot be labeled by Dansyl fluoride, a specific serine-type protein inhibitor. The mutant protein showed no autodegradation in vitro. The mutant hetR gene was introduced into Anabaena strain 884a, a hetR mutant. The resultant strain, Anabaena strain S152A, could not form heterocysts under conditions of nitrogen deprivation even though the up-regulation of the mutant hetR gene was induced upon removal of combined nitrogen. The Anabaena strain 216, which carries a mutant hetR gene encoding S179N HetR and could not form heterocysts, also produced HetR protein upon induction. Sequence comparison shows that Ser152 is conserved in all cyanobacterial HetR. Immunoblotting was used to study HetR induction in both the wild-type and mutant strains. The amount of mutant HetR in strain S152A and in strain 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 might be needed for repression of HetR overproduction under conditions of nitrogen deprivation.     

鱼腥蓝细菌PCC 7120中一个受到NtcA调控的基因alr1390

NtcA是鱼腥蓝细菌中一种重要的氮代谢调控蛋白质,参与异形胞的分化。许多受NtcA调控的基因都至少有一个保守的NtcA结合位点GTA-N8-TAC。经生物信息学分析,鱼腥蓝细菌PCC7120基因alr1390可能的转录起始位点的上游有一个保守的NtcA结合位点GTA-TAGTTTTC-TAC。【目的】为了鉴定alr1390和NtcA之间的关系,【方法】采用实时定量反转录PCR实验(Real-time RT-PCR)和电泳迁移率实验(Electrophoretic mobility shift assays,EMSA)对alr1390和NtcA之间的关系进行了分析。【结果】Real-timeRT-PCR结果显示,alr1390的转录水平在野生型鱼腥蓝细菌PCC7120中缺氮诱导后和诱导前持平,而在ntcA突变体中缺氮诱导12h后呈现上调趋势。但是EMSA实验中没有检测到明显的NtcA和alr1390启动子区片段结合的滞后带,却观察到一条拖带。【结论】这说明alr1390受到NtcA的调控。     

Molecular cloning and characterization of hetR genes from filamentous cyanobacteria

HetR, a serine type protease, plays an important role in heterocyst differentiation in filamentous cyanobacteria. We isolated and sequenced the hetR genes from different heterocystous and filamentous nonheterocystous cyanobacteria. The hetR gene in the heterocyst forming Anabaena variabilis ATCC 29413 FD was interrupted by interposon mutagenesis (mutant strain WSIII8). This mutant does not form heterocysts and shows no diazotrophic growth under aerobic conditions. However, under anaerobic N(2)-fixing conditions, the WSIII8 cells are able to grow, and high nitrogenase (Nif2) activity is detectable. Nif2 expression was demonstrated in each vegetative cell of the filament by immunolocalization 4 h after nitrogen step-down.     

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.     

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.     

The Anabaena sp. strain PCC 7120 asr1734 gene encodes a negative regulator of heterocyst development

The novel asr1734 gene of Anabaena (Nostoc) sp. strain PCC 7120 inhibited heterocyst development when present in extra copies. Overexpression of asr1734 inhibited heterocyst development in several strains including the wild type and two strains that form multiple contiguous heterocysts (Mch phenotype): a PatS null mutant and a hetR(R223W) mutant. Overexpression of asr1734 also caused increased nblA messenger RNA levels, and increased loss of autofluorescence in vegetative cells throughout filaments after nitrogen or sulphur depletion. Unlike the wild type, an asr1734 knockout mutant formed 5% heterocysts after a nitrogen shift from ammonium to nitrate, and formed 15% heterocysts and a weak Mch phenotype after step-down to medium lacking combined nitrogen. After nitrogen step-down, the asr1734 mutant had elevated levels of ntcA messenger RNA. A green fluorescent protein reporter driven by the asr1734 promoter, P(asr1734)-gfp, was expressed specifically in differentiating proheterocysts and heterocysts after nitrogen step-down. Strains overexpressing asr1734 and containing P(hetR)-gfp or P(patS)-gfp reporters failed to show normal patterned upregulation 24 h after nitrogen step-down even though hetR expression was upregulated at 6 h. Apparent orthologues of asr1734 are found only in two other filamentous nitrogen-fixing cyanobacteria, Anabaena variabilis and Nostoc punctiforme.     

Mutagenesis of hetR reveals amino acids necessary for HetR function in the heterocystous cyanobacterium Anabaena sp. strain PCC 7120

HetR is the master regulator of heterocyst differentiation in the filamentous cyanobacterium Anabaena sp. strain PCC 7120. Genetic selection was used to identify 33 amino acid substitutions in HetR that reduced the proportion of cells undergoing heterocyst differentiation to less than 2%. Conservative substitutions in the wild-type HetR protein revealed three mutations that dramatically reduced the amount of heterocyst differentiation when the mutant allele was present in place of the wild-type allele on a replicating plasmid in a mutant lacking hetR on the chromosome. An H69Y substitution resulted in heterocyst formation among less than 0.1% of cells, and D17E and G36A substitutions resulted in a Het- phenotype, compared to heterocyst formation among approximately 25% of cells with the wild-type hetR under the same conditions. The D17E substitution prevented DNA binding activity exhibited by wild-type HetR in mobility shift assays, whereas G36A and H69Y substitutions had no affect on DNA binding. D17E, G36A, and H69Y substitutions also resulted in higher levels of the corresponding HetR protein than of the wild-type protein when each was expressed from an inducible promoter in a hetR deletion strain, suggesting an effect on HetR protein turnover. Surprisingly, C48A and S152A substitutions, which were previously reported to result in a Het- phenotype, were found to have no effect on heterocyst differentiation or patterning when the corresponding mutations were introduced into an otherwise wild-type genetic background in Anabaena sp. strain PCC 7120. The clustering of mutations that satisfied the positive selection near the amino terminus suggests an important role for this part of the protein in HetR function.