Transcriptional dynamics of developmental genes assessed with an FMN-dependent fluorophore in mature heterocysts of Anabaena sp. strain PCC 7120

Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that differentiates nitrogen-fixing heterocysts when available combined nitrogen is limiting. Growth under diazotrophic conditions results in a mixture of ‘new’ (recently differentiated) and ‘old’ (mature) heterocysts. The microoxic environment present in heterocysts makes the interpretation of gene expression using oxygen-dependent fluorophores, including GFP, difficult. The work presented here evaluates the transcriptional dynamics of three developmental genes in mature heterocysts utilizing EcFbFP, a flavin mononucleotide-dependent fluorophore, as the reporter. Expression of both GFP and EcFbFP from the heterologous petE promoter showed that, although GFP and EcFbFP fluoresced in both vegetative cells and new heterocysts, only EcFbFP fluoresced in old heterocysts. A transcriptional fusion of EcFbFP to the late-stage heterocyst-specific nifB promoter displayed continued expression beyond the cessation of GFP fluorescence in heterocysts. Promoter fusions of the master regulator of differentiation, hetR, and its inhibitors, patS and hetN, to GFP and EcFbFP were visualized to determine their role(s) in heterocyst function after morphogenesis. The expression of hetR and hetN was found to persist beyond the completion of development in most heterocysts, whereas patS expression ceased. These data are consistent with a model of heterocyst patterning in which patS is involved in de novo pattern formation, hetN is required for pattern maintenance, and hetR is needed for all stages of development.

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

Life ◽

10.3390/life8040060 ◽

2018 ◽

Vol 8 (4) ◽

pp. 60 ◽

Cited By ~ 5

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.

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

Journal of Bacteriology ◽

10.1128/jb.00776-10 ◽

2010 ◽

Vol 192 (20) ◽

pp. 5526-5533 ◽

Cited By ~ 49

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.

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Anabaena sp. Strain PCC 7120 hetY Gene Influences Heterocyst Development

Journal of Bacteriology ◽

10.1128/jb.185.23.6995-7000.2003 ◽

2003 ◽

Vol 185 (23) ◽

pp. 6995-7000 ◽

Cited By ~ 7

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.

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

Archives of Microbiology ◽

10.1007/s00203-009-0525-4 ◽

2009 ◽

Vol 192 (1) ◽

pp. 23-31 ◽

Cited By ~ 18

Author(s):

Masakazu Toyoshima ◽

Naobumi V. Sasaki ◽

Makoto Fujiwara ◽

Shigeki Ehira ◽

Masayuki Ohmori ◽

...

Keyword(s):

Filamentous Cyanobacterium ◽

Cyanobacterium Anabaena ◽

Anabaena Sp ◽

Pcc 7120

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

Microbiology ◽

10.1099/mic.0.26462-0 ◽

2003 ◽

Vol 149 (11) ◽

pp. 3257-3263 ◽

Cited By ~ 43

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.

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

Frontiers in Microbiology ◽

10.3389/fmicb.2021.793336 ◽

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.

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

Research in Microbiology ◽

10.1016/j.resmic.2020.06.001 ◽

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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Heterocyst-specific flavodiiron protein Flv3B enables oxic diazotrophic growth of the filamentous cyanobacterium Anabaena sp. PCC 7120

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1407327111 ◽

2014 ◽

Vol 111 (30) ◽

pp. 11205-11210 ◽

Cited By ~ 37

Author(s):

M. Ermakova ◽

N. Battchikova ◽

P. Richaud ◽

H. Leino ◽

S. Kosourov ◽

...

Keyword(s):

Filamentous Cyanobacterium ◽

Cyanobacterium Anabaena ◽

Anabaena Sp ◽

Pcc 7120 ◽

Diazotrophic Growth

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Anabaena sp. Strain PCC 7120 Gene devH Is Required for Synthesis of the Heterocyst Glycolipid Layer

Journal of Bacteriology ◽

10.1128/jb.187.7.2326-2331.2005 ◽

2005 ◽

Vol 187 (7) ◽

pp. 2326-2331 ◽

Cited By ~ 32

Author(s):

Martha E. Ramírez ◽

Pratibha B. Hebbar ◽

Ruanbao Zhou ◽

C. Peter Wolk ◽

Stephanie E. Curtis

Keyword(s):

Nitrogen Fixation ◽

Nitrogenase Activity ◽

Regulatory Protein ◽

Protein A ◽

Filamentous Cyanobacterium ◽

Fixed Nitrogen ◽

Ultrastructural Studies ◽

Anabaena Sp ◽

Pcc 7120 ◽

Mutant Forms

ABSTRACT In response to deprivation for fixed nitrogen, the filamentous cyanobacterium Anabaena sp. strain PCC 7120 provides a microoxic intracellular environment for nitrogen fixation through the differentiation of semiregularly spaced vegetative cells into specialized cells called heterocysts. The devH gene is induced during heterocyst development and encodes a product with characteristics of a trans-acting regulatory protein. A devH mutant forms morphologically distinguishable heterocysts but is Fox−, incapable of nitrogen fixation in the presence of oxygen. We demonstrate that rearrangements of nitrogen fixation genes take place normally in the devH mutant and that it is Fix+, i.e., has nitrogenase activity under anoxic conditions. The Fox− phenotype was shown by ultrastructural studies to be associated with the absence of the glycolipid layer of the heterocyst envelope. The expression of glycolipid biosynthetic genes in the mutant is greatly reduced, and heterocyst glycolipids are undetectable.

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