cyAbrB transcriptional regulators as safety devices to inhibit heterocyst differentiation in Anabaena

AbstractCyanobacteria are monophyletic organisms that perform oxygenic photosynthesis. While they exhibit great diversity, they have a common set of genes. However, the essentiality of them for viability has hampered the elucidation of their functions. One example of the genes is cyabrB1 encoding a transcriptional regulator. In the present study, we investigated the function of cyabrB1 in heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 through CRISPR interference, a method we recently utilized for the photosynthetic production of a useful chemical in the strain. Conditional knockdown of cyabrB1 in the presence of nitrate resulted in formation of heterocysts. Two genes, hetP and hepA, which are required for heterocyst formation, were up-regulated by cyabrB1 knockdown in the presence of combined nitrogen sources. The genes are known to be induced by HetR, a master regulator of heterocyst formation. hetR was not induced by cyabrB1 knockdown. hetP and hepA were repressed by direct binding of cyAbrB1 to their promoter regions in a HetR-independent manner. In addition, the over-expression of cyabrB1 abolished heterocyst formation upon nitrogen depletion. Also, knockout of cyabrB2, a paralogue gene of cyabrB1, in addition to cyabrB1 knockdown, enhanced heterocyst formation in the presence of nitrate, suggesting functional redundancy of cyAbrB proteins. We propose that a balance between amounts of HetR and cyAbrB1 is a key factor influencing heterocyst differentiation during nitrogen step-down. cyAbrB proteins are essential safety devices inhibiting heterocyst differentiation.ImportanceSpore formation in Bacillus subtilis and Streptomyces represents non-terminal differentiation and has been extensively studied as models of prokaryotic cell differentiation. In the two organisms, many cells differentiate simultaneously, and the differentiation is governed by a network in which one regulator stands at the top. Differentiation of heterocysts in Anabaena sp. PCC 7120 has also been extensively studied. The differentiation is unique because it is terminal and only 5-10% vegetative cells differentiate into heterocysts. In the present study, we identified cyAbrB1 as a repressor of two genes that are essential for heterocyst formation, hetP and hepA, independent of HetR, which is a master activator for heterocyst differentiation. The finding is reasonable for unique cell differentiation of Anabaena because cyAbrB1 could suppress heterocyst differentiation tightly in vegetative cells, while only cells in which HetR is over-expressed could differentiate into heterocysts.

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cyAbrB Transcriptional Regulators as Safety Devices To Inhibit Heterocyst Differentiation in Anabaena sp. Strain PCC 7120

Journal of Bacteriology ◽

10.1128/jb.00244-19 ◽

2019 ◽

Vol 201 (17) ◽

Cited By ~ 6

Author(s):

Akiyoshi Higo ◽

Eri Nishiyama ◽

Kota Nakamura ◽

Yukako Hihara ◽

Shigeki Ehira

Keyword(s):

Cell Differentiation ◽

Nitrogen Sources ◽

Oxygenic Photosynthesis ◽

Promoter Regions ◽

Independent Manner ◽

Vegetative Cells ◽

Heterocyst Differentiation ◽

Content Type ◽

Safety Devices ◽

Pcc 7120

ABSTRACT Cyanobacteria are monophyletic organisms that perform oxygenic photosynthesis. While they exhibit great diversity, they have a common set of genes. However, the essentiality of them for viability has hampered the elucidation of their functions. One example of these genes is cyabrB1 (also known as calA in Anabaena sp. strain PCC 7120), encoding a transcriptional regulator. In the present study, we investigated the function of calA/cyabrB1 in the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 through CRISPR interference, a method that we recently utilized for the photosynthetic production of a useful chemical in this strain. Conditional knockdown of calA/cyabrB1 in the presence of nitrate resulted in the formation of heterocysts. Two genes, hetP and hepA, which are required for heterocyst formation, were upregulated by calA/cyabrB1 knockdown in the presence of combined nitrogen sources. These genes are known to be induced by HetR, a master regulator of heterocyst formation. hetR was not induced by calA/cyabrB1 knockdown. hetP and hepA were repressed by direct binding of CalA/cyAbrB1 to their promoter regions in a HetR-independent manner. In addition, the overexpression of calA/cyabrB1 abolished heterocyst formation upon nitrogen depletion. Also, knockout of calB/cyabrB2 (a paralogue gene of calA/cyabrB1), in addition to knockdown of calA/cyabrB1, enhanced heterocyst formation in the presence of nitrate, suggesting functional redundancy of cyAbrB proteins. We propose that a balance between amounts of HetR and CalA/cyAbrB1 is a key factor influencing heterocyst differentiation during nitrogen stepdown. We concluded that cyAbrB proteins are essential safety devices that inhibit heterocyst differentiation. IMPORTANCE Spore formation in Bacillus subtilis and Streptomyces has been extensively studied as models of prokaryotic nonterminal cell differentiation. In these organisms, many cells/hyphae differentiate simultaneously, which is governed by a network in which one regulator stands at the top. Differentiation of heterocysts in Anabaena sp. strain PCC 7120 is unique because it is terminal, and only 5 to 10% of vegetative cells differentiate into heterocysts. In this study, we identified CalA/cyAbrB1 as a repressor of two genes that are essential for heterocyst formation independently of HetR, a master activator for heterocyst differentiation. This finding is reasonable for unique cell differentiation of Anabaena because CalA/cyAbrB1 could suppress heterocyst differentiation tightly in vegetative cells, while only cells in which HetR is overexpressed could differentiate into heterocysts.

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

Journal of Bacteriology ◽

10.1128/jb.184.14.3931-3940.2002 ◽

2002 ◽

Vol 184 (14) ◽

pp. 3931-3940 ◽

Cited By ~ 26

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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Specific Glucoside Transporters Influence Septal Structure and Function in the Filamentous, Heterocyst-Forming Cyanobacterium Anabaena sp. Strain PCC 7120

Journal of Bacteriology ◽

10.1128/jb.00876-16 ◽

2017 ◽

Vol 199 (7) ◽

Cited By ~ 13

Author(s):

Mercedes Nieves-Morión ◽

Sigal Lechno-Yossef ◽

Rocío López-Igual ◽

José E. Frías ◽

Vicente Mariscal ◽

...

Keyword(s):

Reducing Power ◽

Structure And Function ◽

Major Facilitator Superfamily ◽

Oxygenic Photosynthesis ◽

Vegetative Cells ◽

Content Type ◽

Combined Nitrogen ◽

Fluorescent Markers ◽

And Function ◽

Pcc 7120

ABSTRACT When deprived of combined nitrogen, some filamentous cyanobacteria contain two cell types: vegetative cells that fix CO2 through oxygenic photosynthesis and heterocysts that are specialized in N2 fixation. In the diazotrophic filament, the vegetative cells provide the heterocysts with reduced carbon (mainly in the form of sucrose) and heterocysts provide the vegetative cells with combined nitrogen. Septal junctions traverse peptidoglycan through structures known as nanopores and appear to mediate intercellular molecular transfer that can be traced with fluorescent markers, including the sucrose analog esculin (a coumarin glucoside) that is incorporated into the cells. Uptake of esculin by the model heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 was inhibited by the α-glucosides sucrose and maltose. Analysis of Anabaena mutants identified components of three glucoside transporters that move esculin into the cells: GlsC (Alr4781) and GlsP (All0261) are an ATP-binding subunit and a permease subunit of two different ABC transporters, respectively, and HepP (All1711) is a major facilitator superfamily (MFS) protein that was shown previously to be involved in formation of the heterocyst envelope. Transfer of fluorescent markers (especially calcein) between vegetative cells of Anabaena was impaired by mutation of glucoside transporter genes. GlsP and HepP interact in bacterial two-hybrid assays with the septal junction-related protein SepJ, and GlsC was found to be necessary for the formation of a normal number of septal peptidoglycan nanopores and for normal subcellular localization of SepJ. Therefore, beyond their possible role in nutrient uptake in Anabaena, glucoside transporters influence the structure and function of septal junctions. IMPORTANCE Heterocyst-forming cyanobacteria have the ability to perform oxygenic photosynthesis and to assimilate atmospheric CO2 and N2. These organisms grow as filaments that fix these gases specifically in vegetative cells and heterocysts, respectively. For the filaments to grow, these types of cells exchange nutrients, including sucrose, which serves as a source of reducing power and of carbon skeletons for the heterocysts. Movement of sucrose between cells in the filament takes place through septal junctions and has been traced with a fluorescent sucrose analog, esculin, that can be taken up by the cells. Here, we identified α-glucoside transporters of Anabaena that mediate uptake of esculin and, notably, influence septal structure and the function of septal junctions.

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HetR-Dependent and -Independent Expression of Heterocyst-Related Genes in an Anabaena Strain Overproducing the NtcA Transcription Factor

Journal of Bacteriology ◽

10.1128/jb.187.6.1985-1991.2005 ◽

2005 ◽

Vol 187 (6) ◽

pp. 1985-1991 ◽

Cited By ~ 34

Author(s):

Elvira Olmedo-Verd ◽

Enrique Flores ◽

Antonia Herrero ◽

Alicia M. Muro-Pastor

Keyword(s):

Transcription Factor ◽

Cell Differentiation ◽

Nitrogen Source ◽

Regulatory Protein ◽

Nitrogen Deficiency ◽

Nitrogen Control ◽

Heterocyst Differentiation ◽

Step Down ◽

Pcc 7120 ◽

Heterocyst Development

ABSTRACT Heterocyst development in the cyanobacterium Anabaena sp. strain PCC 7120 depends on both the global nitrogen control transcription factor NtcA and the cell differentiation regulatory protein HetR, with expression of ntcA and hetR being dependent on each other. In this study we constructed strains that constitutively express the ntcA gene leading to high levels of NtcA protein irrespective of the nitrogen source, and we analyzed the effects of such NtcA levels on heterocyst differentiation. In the NtcA-overproducing strain, heterocyst differentiation, induction of NtcA-dependent heterocyst development genes or operons such as devBCA or the cox2 operon, and NtcA-dependent excision of the 11-kb nifD-intervening element only took place under nitrogen deficiency. Although functional heterocysts were produced in response to nitrogen step-down, the NtcA overproducing strain could not grow diazotrophically. Overexpression of ntcA in a hetR background promoted expression of devBCA in response to ammonium withdrawal and excision of the 11-kb element even in the presence of combined nitrogen. Our results show that some NtcA-dependent heterocyst-related genes can be expressed independently of HetR.

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PatS and Products of Nitrogen Fixation Control Heterocyst Pattern

Journal of Bacteriology ◽

10.1128/jb.183.8.2605-2613.2001 ◽

2001 ◽

Vol 183 (8) ◽

pp. 2605-2613 ◽

Cited By ~ 121

Author(s):

Ho-Sung Yoon ◽

James W. Golden

Keyword(s):

Nitrogen Fixation ◽

Nitrogenase Activity ◽

Early Stage ◽

Critical Role ◽

Filamentous Cyanobacterium ◽

Vegetative Cells ◽

Heterocyst Differentiation ◽

Partial Restoration ◽

Fixation Control ◽

Pcc 7120

ABSTRACT The filamentous cyanobacterium Anabaena sp. strain PCC 7120 forms a developmental pattern of single heterocysts separated by approximately 10 vegetative cells. Heterocysts differentiate from vegetative cells and are specialized for nitrogen fixation. ThepatS gene, which encodes a small peptide that inhibits heterocyst differentiation, is expressed in proheterocysts and plays a critical role in establishing the heterocyst pattern. Here we present further analysis of patS expression and heterocyst pattern formation. A patS-gfp reporter strain revealed clusters of patS-expressing cells during the early stage of heterocyst differentiation. PatS signaling is likely to be involved in the resolution of these clusters. Differentiating cells were inhibited by PatS during the time period 6 to 12 h after heterocyst induction, when groups of differentiating cells were being resolved to a single proheterocyst. Increased transcription ofpatS during development coincided with expression from a new transcription start site. In vegetative cells grown on nitrate, the 5′ end of a transcript for patS was localized 314 bases upstream from the first translation initiation codon. After heterocyst induction, a new transcript with a 5′ end at −39 bases replaced the vegetative cell transcript. A patS mutant grown for several days under nitrogen-fixing conditions showed partial restoration of the normal heterocyst pattern, presumably because of a gradient of nitrogen compounds supplied by the heterocysts. ThepatS mutant formed heterocysts when grown in the presence of nitrate but showed no nitrogenase activity and no obvious heterocyst pattern. We conclude that PatS and products of nitrogen fixation are the main signals determining the heterocyst pattern.

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Proteogenomic Analysis Provides Novel Insight into Genome Annotation and Nitrogen Metabolism in Nostoc sp. PCC 7120

Microbiology Spectrum ◽

10.1128/spectrum.00490-21 ◽

2021 ◽

Author(s):

Shengchao Yu ◽

Mingkun Yang ◽

Jie Xiong ◽

Qi Zhang ◽

Xinxin Gao ◽

...

Keyword(s):

Cell Differentiation ◽

Nitrogen Metabolism ◽

Posttranslational Modifications ◽

Genome Annotation ◽

Vital Role ◽

Oxygenic Photosynthesis ◽

Carbon Cycles ◽

Wide Range ◽

Pcc 7120 ◽

Insight Into

Cyanobacteria are a large group of prokaryotes capable of oxygenic photosynthesis and play a vital role in nitrogen and carbon cycles on Earth. Nostoc 7120 is a commonly used model cyanobacterium for studying cell differentiation and nitrogen metabolism. In this study, we presented the first comprehensive draft map of the Nostoc 7120 proteome and a wide range of posttranslational modifications.

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NtcA-Regulated Heterocyst Differentiation Genes hetC and devB from Anabaena sp. Strain PCC 7120 Exhibit a Similar Tandem Promoter Arrangement

Journal of Bacteriology ◽

10.1128/jb.00616-09 ◽

2009 ◽

Vol 191 (18) ◽

pp. 5765-5774 ◽

Cited By ~ 16

Author(s):

Alicia M. Muro-Pastor ◽

Enrique Flores ◽

Antonia Herrero

Keyword(s):

Promoter Region ◽

Transcriptional Start Site ◽

Short Version ◽

Start Site ◽

Transcription Start ◽

Promoter Regions ◽

Heterocyst Differentiation ◽

Translational Start ◽

Pcc 7120 ◽

Diazotrophic Growth

ABSTRACT Transcription of the hetC gene, whose product is required for heterocyst differentiation, takes place from a long promoter region that includes the previously described HetR-independent, NtcA-activated promoter producing transcripts with a 5′ end corresponding to position −571 with respect to the translational start site of hetC. Northern blot analysis indicated that the accumulation of hetC transcripts depends on HetR, and a second transcriptional start site located at position −293 that leads to NtcA-dependent, HetR-dependent inducible transcription of hetC was identified. Upon nitrogen stepdown, expression of a P hetC ::gfp fusion was transiently induced in specific cells that were differentiating into heterocysts, both when the whole promoter region (containing transcription start points −571 and −293) or a short version (containing only the transcription start point −293) was used. Expression of hetC from the −293 position was delayed in a strain bearing a deleted promoter region lacking sequences upstream from position −570. Such a strain was still able to differentiate functional heterocysts and to grow diazotrophically, although diazotrophic growth was impaired under certain conditions. Similarly, a second, NtcA-dependent, HetR-dependent transcriptional start site was identified at position −454 in the promoter region upstream from the devBCA operon encoding an ABC transport system involved in heterocyst maturation, in which an NtcA-dependent promoter producing transcripts starting at position −704 had been previously noted. Thus, the hetC and devBCA promoter regions exhibit similar tandem promoter arrangements.

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