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

2019 ◽  
Vol 201 (17) ◽  
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.

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

2019 ◽  
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 ◽  
Safety Devices ◽  
Unique Cell ◽  
Pcc 7120

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.

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

2017 ◽  
Vol 199 (7) ◽  
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.

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.

scholarly journals AraR, an l-Arabinose-Responsive Transcriptional Regulator in Corynebacterium glutamicum ATCC 31831, Exerts Different Degrees of Repression Depending on the Location of Its Binding Sites within the Three Target Promoter Regions

2015 ◽  
Vol 197 (24) ◽  
pp. 3788-3796 ◽  
Author(s):  
Takayuki Kuge ◽  
Haruhiko Teramoto ◽  
Masayuki Inui
Keyword(s):  
Corynebacterium Glutamicum ◽  
Binding Sites ◽  
Large Scale ◽  
Transcriptional Regulator ◽  
Scale Production ◽  
Primary Role ◽  
Promoter Regions ◽  
Independent Manner ◽  
Content Type

ABSTRACTInCorynebacterium glutamicumATCC 31831, a LacI-type transcriptional regulator AraR, represses the expression ofl-arabinose catabolism (araBDA), uptake (araE), and the regulator (araR) genes clustered on the chromosome. AraR binds to three sites: one (BSB) between the divergent operons (araBDAandgalM-araR) and two (BSE1and BSE2) upstream ofaraE.l-Arabinose acts as an inducer of the AraR-mediated regulation. Here, we examined the roles of these AraR-binding sites in the expression of the AraR regulon. BSBmutation resulted in derepression of botharaBDAandgalM-araRoperons. The effects of BSE1and/or BSE2mutation onaraEexpression revealed that the two sites independently function as theciselements, but BSE1plays the primary role. However, AraR was shown to bind to these sites with almost the same affinityin vitro. Taken together, the expression ofaraBDAandaraEis strongly repressed by binding of AraR to a single site immediately downstream of the respective transcriptional start sites, whereas the binding site overlapping the −10 or −35 region of thegalM-araRandaraEpromoters is less effective in repression. Furthermore, downregulation ofaraBDAandaraEdependent onl-arabinose catabolism observed in the BSBmutant and the AraR-independentaraRpromoter identified withingalM-araRadd complexity to regulation of the AraR regulon derepressed byl-arabinose.IMPORTANCECorynebacterium glutamicumhas a long history as an industrial workhorse for large-scale production of amino acids. An important aspect of industrial microorganisms is the utilization of the broad range of sugars for cell growth and production process. MostC. glutamicumstrains are unable to use a pentose sugarl-arabinose as a carbon source. However, genes forl-arabinose utilization and its regulation have been recently identified inC. glutamicumATCC 31831. This study elucidates the roles of the multiple binding sites of the transcriptional repressor AraR in the derepression byl-arabinose and thereby highlights the complex regulatory feedback loops in combination withl-arabinose catabolism-dependent repression of the AraR regulon in an AraR-independent manner.

scholarly journals Functional Overlap ofhetPandhetZin Regulation of Heterocyst Differentiation inAnabaenasp. Strain PCC 7120

2018 ◽  
Vol 200 (9) ◽  
pp. e00707-17 ◽  
Author(s):  
He Zhang ◽  
Shuai Wang ◽  
Yali Wang ◽  
Xudong Xu
Keyword(s):  
Nitrogenase Activity ◽  
Terrestrial Ecosystems ◽  
Heterocyst Differentiation ◽  
Content Type ◽  
Reverse Transcription Quantitative Pcr ◽  
Dependent Cell ◽  
Major Factors ◽  
Pcc 7120 ◽  
Relationship Of

ABSTRACTHetR plays a key role in regulation of heterocyst differentiation and patterning inAnabaena. It directly regulates genes involved in heterocyst differentiation (such ashetPandhetZ), genes involved in pattern formation (patA), and many others. In this study, we investigated the functional relationship ofhetPandhetZand their role in HetR-dependent cell differentiation. Coexpression ofhetPandhetZfrom the promoter ofntcA, which encodes the global nitrogen regulator, enabled ahetRmutant to form heterocysts with low aerobic nitrogenase activity. Overexpression ofhetZrestored heterocyst differentiation in ahetPmutant and vice versa. Overexpression ofhetRrestored heterocyst formation in either ahetPor ahetZmutant but not in ahetZ hetPdouble mutant. The functional overlap ofhetPandhetZwas further confirmed by reverse transcription-quantitative PCR (RT-qPCR) and transcriptomic analyses of their effects on gene expression. In addition, yeast two-hybrid and pulldown assays showed the interaction of HetZ with HetR. HetP and HetZ are proposed as the two major factors that control heterocyst formation in response to upregulation ofhetR.IMPORTANCEHeterocyst-forming cyanobacteria contribute significantly to N2fixation in marine, freshwater, and terrestrial ecosystems. Formation of heterocysts enables this group of cyanobacteria to fix N2efficiently under aerobic conditions. HetR, HetP, and HetZ are among the most important factors involved in heterocyst differentiation. We present evidence for the functional overlap ofhetPandhetZand for the key role of the HetR-HetP/HetZ circuit in regulation of heterocyst differentiation. The regulatory mechanism based on HetR, HetP, and HetZ is probably conserved in all heterocyst-forming cyanobacteria.

scholarly journals Three Substrains of the CyanobacteriumAnabaenasp. Strain PCC 7120 Display Divergence in Genomic Sequences andhetCFunction

2018 ◽  
Vol 200 (13) ◽  
Author(s):  
Yali Wang ◽  
Yuan Gao ◽  
Chao Li ◽  
Hong Gao ◽  
Cheng-Cai Zhang ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Single Nucleotide Polymorphisms ◽  
Fluorescent Protein ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Content Type ◽  
Gfp Fluorescence ◽  
Green Fluorescent ◽  
Pcc 7120 ◽  
Heterocyst Development

ABSTRACTAnabaenasp. strain PCC 7120 is a model strain for molecular studies of cell differentiation and patterning in heterocyst-forming cyanobacteria. Subtle differences in heterocyst development have been noticed in different laboratories working on the same organism. In this study, 360 mutations, including single nucleotide polymorphisms (SNPs), small insertion/deletions (indels; 1 to 3 bp), fragment deletions, and transpositions, were identified in the genomes of three substrains. Heterogeneous/heterozygous bases were also identified due to the polyploidy nature of the genome and the multicellular morphology but could be completely segregated when plated after filament fragmentation by sonication.hetCis a gene upregulated in developing cells during heterocyst formation inAnabaenasp. strain PCC 7120 and found in approximately half of other heterocyst-forming cyanobacteria. Inactivation ofhetCin 3 substrains ofAnabaenasp. PCC 7120 led to different phenotypes: the formation of heterocysts, differentiating cells that keep dividing, or the presence of both heterocysts and dividing differentiating cells. The expression of PhetZ-gfpin thesehetCmutants also showed different patterns of green fluorescent protein (GFP) fluorescence. Thus, the function ofhetCis influenced by the genomic background and epistasis and constitutes an example of evolution under way.IMPORTANCEOur knowledge about the molecular genetics of heterocyst formation, an important cell differentiation process for global N2fixation, is mostly based on studies withAnabaenasp. strain PCC 7120. Here, we show that rapid microevolution is under way in this strain, leading to phenotypic variations for certain genes related to heterocyst development, such ashetC. This study provides an example for ongoing microevolution, marked by multiple heterogeneous/heterozygous single nucleotide polymorphisms (SNPs), in a multicellular multicopy-genome microorganism.

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

2005 ◽  
Vol 187 (6) ◽  
pp. 1985-1991 ◽  
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.

scholarly journals PatS and Products of Nitrogen Fixation Control Heterocyst Pattern

2001 ◽  
Vol 183 (8) ◽  
pp. 2605-2613 ◽  
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.

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

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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