scholarly journals HetF and PatA Control Levels of HetR in Anabaena sp. Strain PCC 7120

2008 ◽  
Vol 190 (23) ◽  
pp. 7645-7654 ◽  
Author(s):  
Douglas D. Risser ◽  
Sean M. Callahan
Keyword(s):  
Active Site ◽  
Transcriptional Activity ◽  
Inverse Correlation ◽  
Inducible Promoter ◽  
Site Directed Mutagenesis ◽  
Filamentous Cyanobacterium ◽  
Transcription Start ◽  
Anabaena Sp ◽  
Pcc 7120 ◽  
Active Site Histidine

ABSTRACT Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that differentiates heterocysts in response to deprivation of combined nitrogen. A hetF deletion strain lacked heterocysts and had aberrant cell morphology. Site-directed mutagenesis of the predicted active-site histidine and cysteine residues of this putative caspase-hemoglobinase fold protease abolished HetF function, supporting the hypothesis that HetF is a protease. Deletion of patA, which is necessary for the formation of most intercalary heterocysts, or hetF resulted in an increase in HetR protein, and extra copies of hetF on a plasmid functionally bypassed the deletion of patA. A hetR-gfp translational fusion expressed from an inducible promoter demonstrated that hetF-dependent downregulation of HetR levels occurs rapidly in vegetative cells, as well as developing heterocysts. “Mosaic” filaments in which only one cell of a filament had a copy of hetR or hetF indicated that hetF is required for differentiation only in cells that will become heterocysts. hetF was required for transcription from a hetR-dependent transcription start point of the hetR promoter and induction of transcription from the patS promoter. The inverse correlation between the level of HetR protein and transcription from hetR-dependent promoters suggests that the transcriptional activity of HetR is regulated by HetF and PatA.

scholarly journals Evaluation of inducible promoter–riboswitch constructs for heterologous protein expression in the cyanobacterial species Anabaena sp. PCC 7120

2021 ◽  
Author(s):  
Jessee Svoboda ◽  
Brenda Cisneros ◽  
Benjamin Philmus
Keyword(s):  
Synthetic Biology ◽  
Inducible Promoter ◽  
Model Organisms ◽  
Heterologous Protein Expression ◽  
Filamentous Cyanobacterium ◽  
Vitro Assay ◽  
Anabaena Sp ◽  
The Difference ◽  
Pcc 7120

Abstract Cyanobacteria are promising chassis for synthetic biology applications due to the fact that they are photosynthetic organisms capable of growing in simple, inexpensive media. Given their slower growth rate than other model organisms such as Escherichia coli and Saccharomyces cerevisiae, there are fewer synthetic biology tools and promoters available for use in model cyanobacteria. Here, we compared a small library of promoter–riboswitch constructs for synthetic biology applications in Anabaena sp. PCC 7120, a model filamentous cyanobacterium. These constructs were designed from six cyanobacterial promoters of various strengths, each paired with one of two theophylline-responsive riboswitches. The promoter–riboswitch pairs were cloned upstream of a chloramphenicol acetyltransferase (cat) gene, and CAT activity was quantified using an in vitro assay. Addition of theophylline to cultures increased the CAT activity in almost all cases, allowing inducible protein production with natively constitutive promoters. We found that riboswitch F tended to have a lower induced and uninduced production compared to riboswitch E for the weak and medium promoters, although the difference was larger for the uninduced production, in accord with previous research. The strong promoters yielded a higher baseline CAT activity than medium strength and weak promoters. In addition, we observed no appreciable difference between CAT activity measured from strong promoters cultured in uninduced and induced conditions. The results of this study add to the genetic toolbox for cyanobacteria and allow future natural product and synthetic biology researchers to choose a construct that fits their needs.

scholarly journals Site-Directed Mutagenesis of the Anabaena sp. Strain PCC 7120 Nitrogenase Active Site To Increase Photobiological Hydrogen Production

2010 ◽  
Vol 76 (20) ◽  
pp. 6741-6750 ◽  
Author(s):  
Hajime Masukawa ◽  
Kazuhito Inoue ◽  
Hidehiro Sakurai ◽  
C. Peter Wolk ◽  
Robert P. Hausinger
Keyword(s):  
Active Site ◽  
Electron Flow ◽  
Hydrogen Gas ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Proton Reduction ◽  
Anabaena Sp ◽  
Pcc 7120 ◽  
Iron Molybdenum Cofactor

ABSTRACT Cyanobacteria use sunlight and water to produce hydrogen gas (H2), which is potentially useful as a clean and renewable biofuel. Photobiological H2 arises primarily as an inevitable by-product of N2 fixation by nitrogenase, an oxygen-labile enzyme typically containing an iron-molybdenum cofactor (FeMo-co) active site. In Anabaena sp. strain 7120, the enzyme is localized to the microaerobic environment of heterocysts, a highly differentiated subset of the filamentous cells. In an effort to increase H2 production by this strain, six nitrogenase amino acid residues predicted to reside within 5 � of the FeMo-co were mutated in an attempt to direct electron flow selectively toward proton reduction in the presence of N2. Most of the 49 variants examined were deficient in N2-fixing growth and exhibited decreases in their in vivo rates of acetylene reduction. Of greater interest, several variants examined under an N2 atmosphere significantly increased their in vivo rates of H2 production, approximating rates equivalent to those under an Ar atmosphere, and accumulated high levels of H2 compared to the reference strains. These results demonstrate the feasibility of engineering cyanobacterial strains for enhanced photobiological production of H2 in an aerobic, nitrogen-containing environment.

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 Inactivation of a Heterocyst-Specific Invertase Indicates a Principal Role of Sucrose Catabolism in Heterocysts of Anabaena sp.

2010 ◽  
Vol 192 (20) ◽  
pp. 5526-5533 ◽  
Author(s):  
Rocío López-Igual ◽  
Enrique Flores ◽  
Antonia Herrero
Keyword(s):  
Fluorescent Protein ◽  
Northern Analysis ◽  
Filamentous Cyanobacterium ◽  
Nitrogen Deprivation ◽  
Vegetative Cells ◽  
Anabaena Sp ◽  
Green Fluorescent ◽  
Pcc 7120 ◽  
Diazotrophic Growth

ABSTRACT Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that carries out N2 fixation in specialized cells called heterocysts, which exchange nutrients and regulators with the filament's vegetative cells that perform the photosynthetic fixation of CO2. The Anabaena genome carries two genes coding for alkaline/neutral invertases, invA and invB. As shown by Northern analysis, both genes were expressed monocistronically and induced under nitrogen deprivation, although induction was stronger for invB than for invA. Whereas expression of an InvA-N-GFP fusion (green fluorescent protein [GFP] fused to the N terminus of the InvA protein [InvA-N]) was homogeneous along the cyanobacterial filament, consistent with the lack of dependence on HetR, expression of an InvB-N-GFP fusion upon combined nitrogen deprivation took place mainly in differentiating and mature heterocysts. In an hetR genetic background, the InvB-N-GFP fusion was strongly expressed all along the filament. An insertional mutant of invA could grow diazotrophically but was impaired in nifHDK induction and exhibited an increased frequency of heterocysts, suggesting a regulatory role of the invertase-mediated carbon flux in vegetative cells. In contrast, an invB mutant was strongly impaired in diazotrophic growth, showing a crucial role of sucrose catabolism mediated by the InvB invertase in the heterocysts.

scholarly journals Anabaena sp. Strain PCC 7120 hetY Gene Influences Heterocyst Development

2003 ◽  
Vol 185 (23) ◽  
pp. 6995-7000 ◽  
Author(s):  
Ho-Sung Yoon ◽  
Martin H. Lee ◽  
Jin Xiong ◽  
James W. Golden
Keyword(s):  
Atp Binding ◽  
Binding Motif ◽  
Filamentous Cyanobacterium ◽  
Hypothetical Proteins ◽  
Nitrogen Fixing ◽  
Fixed Nitrogen ◽  
Anabaena Sp ◽  
Time Required ◽  
Pcc 7120 ◽  
Heterocyst Development

ABSTRACT The filamentous cyanobacterium Anabaena (Nostoc) sp. strain PCC 7120 responds to starvation for fixed nitrogen by producing a semiregular pattern of nitrogen-fixing cells called heterocysts. Overexpression of the hetY gene partially suppressed heterocyst formation, resulting in an abnormal heterocyst pattern. Inactivation of hetY increased the time required for heterocyst maturation and caused defects in heterocyst morphology. The 489-bp hetY gene (alr2300), which is adjacent to patS (asl2301), encodes a protein that belongs to a conserved family of bacterial hypothetical proteins that contain an ATP-binding motif.

Early candidacy for differentiation into heterocysts in the filamentous cyanobacterium Anabaena sp. PCC 7120

2009 ◽  
Vol 192 (1) ◽  
pp. 23-31 ◽  
Author(s):  
Masakazu Toyoshima ◽  
Naobumi V. Sasaki ◽  
Makoto Fujiwara ◽  
Shigeki Ehira ◽  
Masayuki Ohmori ◽  
...  
Keyword(s):  
Filamentous Cyanobacterium ◽  
Cyanobacterium Anabaena ◽  
Anabaena Sp ◽  
Pcc 7120

An increase in the level of 2-oxoglutarate promotes heterocyst development in the cyanobacterium Anabaena sp. strain PCC 7120

Microbiology ◽  
2003 ◽  
Vol 149 (11) ◽  
pp. 3257-3263 ◽  
Author(s):  
Jian-Hong Li ◽  
Sophie Laurent ◽  
Viren Konde ◽  
Sylvie Bédu ◽  
Cheng-Cai Zhang
Keyword(s):  
Inorganic Nitrogen ◽  
Cell Types ◽  
Filamentous Cyanobacterium ◽  
Nitrogen Status ◽  
Gene Encoding ◽  
Combined Nitrogen ◽  
Cyanobacterium Anabaena ◽  
Anabaena Sp ◽  
Pcc 7120 ◽  
Heterocyst Development

In the filamentous cyanobacterium Anabaena sp. strain PCC 7120, a starvation of combined nitrogen induces differentiation of heterocysts, cells specialized in nitrogen fixation. How do filaments perceive the limitation of the source of combined nitrogen, and what determines the proportion of heterocysts? In cyanobacteria, 2-oxoglutarate provides a carbon skeleton for the incorporation of inorganic nitrogen. Recently, it has been proposed that the concentration of 2-oxoglutarate reflects the nitrogen status in cyanobacteria. To investigate the effect of 2-oxoglutarate on heterocyst development, a heterologous gene encoding a 2-oxoglutarate permease under the control of a regulated promoter was expressed in Anabaena sp. PCC 7120. The increase of 2-oxoglutarate within cells can trigger heterocyst differentiation in a subpopulation of filaments even in the presence of nitrate. In the absence of a source of combined nitrogen, it can increase heterocyst frequency, advance the timing of commitment to heterocyst development and further increase the proportion of heterocysts in a patS mutant. Here, it is proposed that the intracellular concentration of 2-oxoglutarate is involved in the determination of the proportion of the two cell types according to the carbon/nitrogen status of the filament.

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.

Function analysis of RNase E in the filamentous cyanobacterium Anabaena sp. PCC 7120

2020 ◽  
Vol 171 (5-6) ◽  
pp. 194-202
Author(s):  
Huaduo Yan ◽  
Yarui Cheng ◽  
Li Wang ◽  
Wenli Chen
Keyword(s):  
Function Analysis ◽  
Filamentous Cyanobacterium ◽  
Rnase E ◽  
Cyanobacterium Anabaena ◽  
Anabaena Sp ◽  
Pcc 7120
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