scholarly journals NrrA Directly Regulates Expression of hetR during Heterocyst Differentiation in the Cyanobacterium Anabaena sp. Strain PCC 7120

2006 ◽  
Vol 188 (24) ◽  
pp. 8520-8525 ◽  
Author(s):  
Shigeki Ehira ◽  
Masayuki Ohmori
Keyword(s):  
Transcription Initiation ◽  
Response Regulator ◽  
Nitrogen Deprivation ◽  
Heterocyst Differentiation ◽  
Combined Nitrogen ◽  
Regulatory Cascade ◽  
Cyanobacterium Anabaena ◽  
Anabaena Sp ◽  
Pcc 7120

ABSTRACT Heterocyst differentiation in the cyanobacterium Anabaena sp. strain PCC 7120 requires NtcA, the global nitrogen regulator in cyanobacteria, and HetR, the master regulator of heterocyst differentiation. Expression of hetR is upregulated by nitrogen deprivation, and its upregulation depends on NtcA. However, it has not yet been revealed how NtcA regulates the expression of hetR. In the experiments presented here, it was confirmed that NrrA (All4312), a nitrogen-responsive response regulator, was required for the upregulation of hetR. The use of the nitrogen-responsive transcription initiation sites (TISs) for the hetR gene depended upon NrrA. NrrA bound specifically to the region upstream of TISs located at positions −728 and −696 in vitro. Overexpression of nrrA resulted in enhanced hetR expression and heterocyst formation. A molecular regulatory cascade is proposed whereby NtcA upregulates the expression of nrrA upon limitation of combined nitrogen in the medium and then NrrA upregulates the expression of hetR, leading to heterocyst differentiation.

scholarly journals c-di-GMP Homeostasis Is Critical for Heterocyst Development in Anabaena sp. PCC 7120

2021 ◽  
Vol 12 ◽  
Author(s):  
Min Huang ◽  
Ju-Yuan Zhang ◽  
Xiaoli Zeng ◽  
Cheng-Cai Zhang
Keyword(s):  
Nitrogen Starvation ◽  
Filamentous Cyanobacterium ◽  
Nitrogen Deprivation ◽  
Heterocyst Differentiation ◽  
Combined Nitrogen ◽  
Heterocyst Frequency ◽  
Genes Encoding ◽  
Anabaena Sp ◽  
Opposing Effects ◽  
Pcc 7120

c-di-GMP is a ubiquitous bacterial signal regulating various physiological process. Anabaena PCC 7120 (Anabaena) is a filamentous cyanobacterium able to form regularly-spaced heterocysts for nitrogen fixation, in response to combined-nitrogen deprivation in 24h. Anabaena possesses 16 genes encoding proteins for c-di-GMP metabolism, and their functions are poorly characterized, except all2874 (cdgS) whose deletion causes a decrease in heterocyst frequency 48h after nitrogen starvation. We demonstrated here that c-di-GMP levels increased significantly in Anabaena after combined-nitrogen starvation. By inactivating each of the 16 genes, we found that the deletion of all1175 (cdgSH) led to an increase of heterocyst frequency 24h after nitrogen stepdown. A double mutant ΔcdgSHΔcdgS had an additive effect over the single mutants in regulating heterocyst frequency, indicating that the two genes acted at different time points for heterocyst spacing. Biochemical and genetic data further showed that the functions of CdgSH and CdgS in the setup or maintenance of heterocyst frequency depended on their opposing effects on the intracellular levels of c-di-GMP. Finally, we demonstrated that heterocyst differentiation was completely inhibited when c-di-GMP levels became too high or too low. Together, these results indicate that the homeostasis of c-di-GMP level is important for heterocyst differentiation in Anabaena.

scholarly journals Identification of the Active Site of HetR Protease and Its Requirement for Heterocyst Differentiation in the Cyanobacterium Anabaena sp. Strain PCC 7120

2000 ◽  
Vol 182 (6) ◽  
pp. 1575-1579 ◽  
Author(s):  
Yuqing Dong ◽  
Xu Huang ◽  
Xiang-Yu Wu ◽  
Jindong Zhao
Keyword(s):  
Active Site ◽  
Maximum Level ◽  
Mutant Protein ◽  
Nitrogen Deprivation ◽  
Wild Type ◽  
Heterocyst Differentiation ◽  
Cyanobacterium Anabaena ◽  
Step Down ◽  
Pcc 7120

ABSTRACT 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 intoAnabaena 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.

scholarly journals The Ser/Thr Kinase PknH Is Essential for Maintaining Heterocyst Pattern in the Cyanobacterium Anabaena sp. Strain PCC 7120

Life ◽  
2018 ◽  
Vol 8 (3) ◽  
pp. 34 ◽  
Author(s):  
Shun-ichi Fukushima ◽  
Shigeki Ehira
Keyword(s):  
Pattern Formation ◽  
Lateral Inhibition ◽  
Time Lapse ◽  
Filamentous Cyanobacterium ◽  
Nitrogen Deprivation ◽  
Heterocyst Differentiation ◽  
Cyanobacterium Anabaena ◽  
Reporter Strains ◽  
Anabaena Sp ◽  
Pcc 7120

In the filamentous cyanobacterium Anabaena sp. strain, PCC 7120, heterocysts (which are nitrogen-fixing cells) are formed in the absence of combined nitrogen in the medium. Heterocysts are separated from one another by 10 to 15 vegetative cells along the filaments, which consist of a few hundred of cells. hetR is necessary for heterocyst differentiation; and patS and hetN, expressed in heterocysts, play important roles in heterocyst pattern formation by laterally inhibiting the expression of hetR in adjacent cells. The results of this study indicated that pknH, which encodes a Ser/Thr kinase, was also involved in heterocyst pattern formation. In the pknH mutant, the heterocyst pattern was normal within 24 h after nitrogen deprivation, but multiple contiguous heterocysts were formed from 24 to 48 h. A time-lapse analysis of reporter strains harboring a fusion between gfp and the hetR promoter indicated that pknH was required to suppress hetR expression in cells adjacent to the preexisting heterocysts. These results indicated that pknH was necessary for the lateral inhibition of heterocyst differentiation to maintain the heterocyst pattern.

scholarly journals Transcription Activation by NtcA and 2-Oxoglutarate of Three Genes Involved in Heterocyst Differentiation in the Cyanobacterium Anabaena sp. Strain PCC 7120

2008 ◽  
Vol 190 (18) ◽  
pp. 6126-6133 ◽  
Author(s):  
Ana Valladares ◽  
Enrique Flores ◽  
Antonia Herrero
Keyword(s):  
Rna Polymerase ◽  
Transcription Activation ◽  
Nitrogen Deprivation ◽  
Stringent Requirement ◽  
Heterocyst Differentiation ◽  
Cyanobacterium Anabaena ◽  
Polymerase Binding ◽  
Pcc 7120 ◽  
Positive Effect

ABSTRACT In Anabaena sp. strain PCC 7120, differentiation of heterocysts takes place in response to the external cue of combined nitrogen deprivation, allowing the organism to fix atmospheric nitrogen in oxic environments. NtcA, a global transcriptional regulator of cyanobacteria, is required for activation of the expression of multiple genes involved in heterocyst differentiation, including key regulators that are specific to the process. We have set up a fully defined in vitro system, which includes the purified Anabaena RNA polymerase, and have studied the effects of NtcA and its signaling effector 2-oxoglutarate on RNA polymerase binding, open complex formation, and transcript production from promoters of the hetC, nrrA, and devB genes that are activated by NtcA at different stages of heterocyst differentiation. Both RNA polymerase and NtcA could specifically bind to the target DNA in the absence of any effector. 2-Oxoglutarate had a moderate positive effect on NtcA binding, and NtcA had a limited positive effect on RNA polymerase recruitment at the promoters. However, a stringent requirement of both NtcA and 2-oxoglutarate was observed for the detection of open complexes and transcript production at the three investigated promoters. These results support a key role for 2-oxoglutarate in transcription activation in the developing heterocyst.

scholarly journals NrrA directly regulates expression of the fraF gene and antisense RNAs for fraE in the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120

Microbiology ◽  
2014 ◽  
Vol 160 (5) ◽  
pp. 844-850 ◽  
Author(s):  
Shigeki Ehira ◽  
Masayuki Ohmori
Keyword(s):  
Response Regulator ◽  
Repeat Sequence ◽  
Nitrogen Deprivation ◽  
Antisense Rnas ◽  
Regulated Expression ◽  
Cyanobacterium Anabaena ◽  
Anabaena Sp ◽  
Dna Strand ◽  
Pcc 7120 ◽  
Diazotrophic Growth

The heterocystous cyanobacterium Anabaena sp. strain PCC 7120 grows as linear multicellular filaments that can contain hundreds of cells. Heterocysts, which are specialized cells for nitrogen fixation, are regularly intercalated among photosynthetic vegetative cells, and these cells are metabolically dependent on each other. Thus, multicellularity is essential for diazotrophic growth of heterocystous cyanobacteria. In Anabaena sp. strain PCC 7120, the fraF gene, which is required to limit filament length, is induced by nitrogen deprivation. The fraF transcripts extend to the fraE gene, which lies on the opposite DNA strand and could possess dual functionality, mRNAs for fraF and antisense RNAs for fraE. In the present study, we found that NrrA, a nitrogen-regulated response regulator, directly regulated expression of fraF. Induction of fraF by nitrogen deprivation was abolished by the nrrA disruption. NrrA specifically bound to the promoter region of fraF, and recognized an inverted repeat sequence. Thus, it is concluded that NrrA controls expression of mRNAs for fraF and antisense RNAs for fraE in response to nitrogen deprivation.

scholarly journals All1371 is a polyphosphate-dependent glucokinase in Anabaena sp. PCC 7120

Microbiology ◽  
2014 ◽  
Vol 160 (12) ◽  
pp. 2807-2819 ◽  
Author(s):  
Friederike Klemke ◽  
Gabriele Beyer ◽  
Linda Sawade ◽  
Ali Saitov ◽  
Thomas Korte ◽  
...  
Keyword(s):  
Divalent Cations ◽  
Enzymic Activity ◽  
Phosphoryl Group ◽  
Nitrogen Deprivation ◽  
Ping Pong ◽  
Nucleoside Triphosphates ◽  
Anabaena Sp ◽  
Pcc 7120 ◽  
Promoter Studies

The polyphosphate glucokinases can phosphorylate glucose to glucose 6-phosphate using polyphosphate as the substrate. ORF all1371 encodes a putative polyphosphate glucokinase in the filamentous heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. Here, ORF all1371 was heterologously expressed in Escherichia coli, and its purified product was characterized. Enzyme activity assays revealed that All1371 is an active polyphosphate glucokinase that can phosphorylate both glucose and mannose in the presence of divalent cations in vitro. Unlike many other polyphosphate glucokinases, for which nucleoside triphosphates (e.g. ATP or GTP) act as phosphoryl group donors, All1371 required polyphosphate to confer its enzymic activity. The enzymic reaction catalysed by All1371 followed classical Michaelis–Menten kinetics, with k cat = 48.2 s−1 at pH 7.5 and 28 °C and K M = 1.76 µM and 0.118 mM for polyphosphate and glucose, respectively. Its reaction mechanism was identified as a particular multi-substrate mechanism called the ‘bi-bi ping-pong mechanism’. Bioinformatic analyses revealed numerous polyphosphate-dependent glucokinases in heterocyst-forming cyanobacteria. Viability of an Anabaena sp. PCC 7120 mutant strain lacking all1371 was impaired under nitrogen-fixing conditions. GFP promoter studies indicate expression of all1371 under combined nitrogen deprivation. All1371 might play a substantial role in Anabaena sp. PCC 7120 under these conditions.

scholarly journals Manipulation of Pattern of Cell Differentiation in a hetR Mutant of Anabaena sp. PCC 7120 by Overexpressing hetZ Alone or with hetP

Life ◽  
2018 ◽  
Vol 8 (4) ◽  
pp. 60 ◽  
Author(s):  
He Zhang ◽  
Xudong Xu
Keyword(s):  
Cell Differentiation ◽  
Peptide Inhibitor ◽  
Filamentous Cyanobacterium ◽  
Heterocyst Differentiation ◽  
Cyanobacterium Anabaena ◽  
Anabaena Sp ◽  
Pcc 7120

In the filamentous cyanobacterium, Anabaena sp. PCC 7120, single heterocysts differentiate at semi-regular intervals in response to nitrogen stepdown. HetR is a principal regulator of heterocyst differentiation, and hetP and hetZ are two genes that are regulated directly by HetR. In a hetR mutant generated from the IHB (Institute of Hydrobiology) substrain of PCC 7120, heterocyst formation can be restored by moderate expression of hetZ and hetP. The resulting heterocysts are located at terminal positions. We used a tandem promoter, PrbcLPpetE, to express hetZ and hetP strongly in the hetR mutant. Co-expression of hetZ and hetP enabled the hetR mutant to form multiple contiguous heterocysts at both terminal and intercalary positions. Expression of hetZ, alone resulted in terminally located heterocysts, whereas expression of hetP, alone produced enlarged cells in strings. In the absence of HetR, formation of heterocysts was insensitive to the peptide inhibitor, RGSGR.

scholarly journals Novel DNA-Binding Proteins in the Cyanobacterium Anabaena sp. Strain PCC 7120

2002 ◽  
Vol 184 (14) ◽  
pp. 3931-3940 ◽  
Author(s):  
Olga A. Koksharova ◽  
C. Peter Wolk
Keyword(s):  
Dna Binding ◽  
Insertional Mutagenesis ◽  
Binding Proteins ◽  
Dna Binding Proteins ◽  
Vegetative Cells ◽  
Heterocyst Differentiation ◽  
The Third ◽  
Cyanobacterium Anabaena ◽  
Anabaena Sp ◽  
Pcc 7120

ABSTRACT As an approach towards elucidation of the biochemical regulation of the progression of heterocyst differentiation in Anabaena sp. strain PCC 7120, we have identified proteins that bind to a 150-bp sequence upstream from hepC, a gene that plays a role in the synthesis of heterocyst envelope polysaccharide. Such proteins were purified in four steps from extracts of vegetative cells of Anabaena sp. Two of these proteins (Abp1 and Abp2) are encoded by neighboring genes in the Anabaena sp. chromosome. The genes that encode the third (Abp3) and fourth (Abp4) proteins are situated at two other loci in that chromosome. Insertional mutagenesis of abp2 and abp3 blocked expression of hepC and hepA and prevented heterocyst maturation and aerobic fixation of N2.

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