scholarly journals Heterocyst Development and Diazotrophic Metabolism in Terminal Respiratory Oxidase Mutants of the Cyanobacterium Anabaena sp. Strain PCC 7120

2007 ◽  
Vol 189 (12) ◽  
pp. 4425-4430 ◽  
Author(s):  
Ana Valladares ◽  
Iris Maldener ◽  
Alicia M. Muro-Pastor ◽  
Enrique Flores ◽  
Antonia Herrero
Keyword(s):  
Nitrogenase Activity ◽  
Nitrogen Deprivation ◽  
Wild Type ◽  
Cyanobacterium Anabaena ◽  
In The Wild ◽  
Membrane Reorganization ◽  
Respiratory Oxidases ◽  
And Performance ◽  
Pcc 7120 ◽  
Heterocyst Development

ABSTRACT Heterocyst development was analyzed in mutants of the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 bearing inactivated cox2 and/or cox3 genes, encoding heterocyst-specific terminal respiratory oxidases. At the morphological level, the cox2 cox3 double mutant (strain CSAV141) was impaired in membrane reorganization involving the so-called honeycomb system that in the wild-type strain is largely or exclusively devoted to respiration, accumulated glycogen granules at conspicuously higher levels than the wild type (in both vegetative cells and heterocysts), and showed a delay in carboxysome degradation upon combined nitrogen deprivation. Consistently, chemical analysis confirmed higher accumulation of glycogen in strain CSAV141 than in the wild type. No impairment was observed in the formation of the glycolipid or polysaccharide layers of the heterocyst envelope, consistent with the chemical detection of heterocyst-specific glycolipids, or in the expression of the heterocyst-specific genes nifHDK and fdxH. However, nitrogenase activity under oxic conditions was impaired in strain CSAV135 (cox3) and undetectable in strain CSAV141 (cox2 cox3). These results show that these dedicated oxidases are required for normal development and performance of the heterocysts and indicate a central role of Cox2 and, especially, of Cox3 in the respiratory activity of the heterocysts, decisively contributing to protection of the N2 fixation machinery against oxygen. However, in contrast to the case for other diazotrophic bacteria, expression of nif genes in Anabaena seems not to be affected by oxygen.

scholarly journals PII Is Important in Regulation of Nitrogen Metabolism but Not Required for Heterocyst Formation in the Cyanobacterium Anabaena sp. PCC 7120

2007 ◽  
Vol 282 (46) ◽  
pp. 33641-33648 ◽  
Author(s):  
Ying Zhang ◽  
Hai Pu ◽  
Qingsong Wang ◽  
Shu Cheng ◽  
Weixing Zhao ◽  
...  
Keyword(s):  
Nitrogen Metabolism ◽  
Nitrate Uptake ◽  
Nitrogenase Activity ◽  
Tyrosine Nitration ◽  
Wild Type ◽  
Cyanobacterium Anabaena ◽  
In The Wild ◽  
Anabaena Sp ◽  
Key Residues ◽  
Pcc 7120

PII is an important signal protein for regulation of nitrogen metabolism in bacteria and plants. We constructed a mutant of glnB, encoding PII, in a heterocystous cyanobacterium, Anabaena sp. PCC 7120, with a cre-loxP system. The mutant (MP2α) grew more slowly than the wild type under all nitrogen regimens. It excreted a large amount of ammonium when grown on nitrate due to altered activities of glutamine synthetase and nitrate reductase. MP2α had a low nitrogenase activity but was able to form heterocysts under diazotrophic conditions, suggesting that PII is not required for heterocyst differentiation. Analysis of the PII with mass spectroscopy found tyrosine nitration at Tyr-51 under diazotrophic conditions while no phosphorylation at Ser-49 was detected. The strains 51F and 49A, which have PII with mutations of Y51F and S49A, respectively, were constructed to analyze the functions of the two key residues on the T-loop. Like MP2α, they had low nitrogenase activity and grew slowly under diazotrophic conditions. 49A was also impaired in nitrate uptake and formed heterocysts in the presence of nitrate. The up-regulation of ntcA after nitrogen step-down, which was present in the wild type, was not observed in 51F and 49A. While our results showed that the Ser-49 residue is important to the function of PII in Anabaena sp. PCC 7120, evidence from the PII pattern of the wild type and 49A in non-denaturing gel electrophoresis suggested that Ser-49 is not modified. The possible physiological roles of tyrosine nitration of PII are discussed.

scholarly journals pbpB, a Gene Coding for a Putative Penicillin-Binding Protein, Is Required for Aerobic Nitrogen Fixation in the Cyanobacterium Anabaena sp. Strain PCC7120

2001 ◽  
Vol 183 (2) ◽  
pp. 628-636 ◽  
Author(s):  
Sara Lázaro ◽  
Francisca Fernández-Piñas ◽  
Eduardo Fernández-Valiente ◽  
Amaya Blanco-Rivero ◽  
Francisco Leganés
Keyword(s):  
Sequence Similarity ◽  
Shuttle Vector ◽  
Molecular Size ◽  
Nitrogen Deprivation ◽  
Wild Type ◽  
Reading Frame ◽  
Cyanobacterium Anabaena ◽  
In The Wild ◽  
Peptidoglycan Layer

ABSTRACT Transposon mutagenesis of Anabaena sp. strain PCC7120 led to the isolation of a mutant strain, SNa1, which is unable to fix nitrogen aerobically but is perfectly able to grow with combined nitrogen (i.e., nitrate). Reconstruction of the transposon mutation of SNa1 in the wild-type strain reproduced the phenotype of the original mutant. The transposon had inserted within an open reading frame whose translation product shows significant homology with a family of proteins known as high-molecular-weight penicillin-binding proteins (PBPs), which are involved in the synthesis of the peptidoglycan layer of the cell wall. A sequence similarity search allowed us to identify at least 12 putative PBPs in the recently sequencedAnabaena sp. strain PCC7120 genome, which we have named and organized according to predicted molecular size and theEscherichia coli nomenclature for PBPs; based on this nomenclature, we have denoted the gene interrupted in SNal aspbpB and its product as PBP2. The wild-type form ofpbpB on a shuttle vector successfully complemented the mutation in SNa1. In vivo expression studies indicated that PBP2 is probably present when both sources of nitrogen, nitrate and N2, are used. When nitrate is used, the function of PBP2 either is dispensable or may be substituted by other PBPs; however, under nitrogen deprivation, where the differentiation of the heterocyst takes place, the role of PBP2 in the formation and/or maintenance of the peptidoglycan layer is essential.

scholarly journals hetL Overexpression Stimulates Heterocyst Formation in Anabaena sp. Strain PCC 7120

2002 ◽  
Vol 184 (24) ◽  
pp. 6873-6881 ◽  
Author(s):  
Duan Liu ◽  
James W. Golden
Keyword(s):  
Null Mutant ◽  
Wild Type ◽  
Heterologous Promoter ◽  
Repeat Proteins ◽  
Overexpression Strain ◽  
In The Wild ◽  
Anabaena Sp ◽  
Pcc 7120 ◽  
Diazotrophic Growth ◽  
Heterocyst Development

ABSTRACT The cyanobacterium Anabaena sp. strain PCC 7120 forms single heterocysts about every 10 to 15 vegetative cells along filaments. PatS is thought to be a peptide intercellular signal made by developing heterocysts that prevents neighboring cells from differentiating. Overexpression of the patS gene suppresses heterocyst formation. The hetL gene (all3740) was isolated in a genetic screen to identify genes involved in PatS signaling. Extracopy hetL allowed heterocyst formation in a patS overexpression strain. hetL overexpression from a heterologous promoter in wild-type Anabaena PCC 7120 induced multiple-contiguous heterocysts (Mch) in nitrate-containing medium. The predicted HetL protein is composed almost entirely of pentapeptide repeats with a consensus of A(D/N)L*X, where * is a polar amino acid. Thirty Anabaena PCC 7120 genes contain this repeat motif. A synthetic pentapeptide corresponding to the last 5 amino acids of PatS, which suppresses heterocyst formation in the wild type, did not suppress heterocyst formation in a hetL overexpression strain, indicating that HetL overexpression is affecting heterocyst regulation downstream of PatS production. The transcription regulator NtcA is required for the initiation of heterocyst formation. hetL overexpression allowed the initiation of heterocyst development in an ntcA-null mutant, but differentiation was incomplete. hetR and hetC mutations that block heterocyst development are epistatic to hetL overexpression. A hetL-null mutant showed normal heterocyst development and diazotrophic growth, which could indicate that it is not normally involved in regulating development, that it normally plays a nonessential accessory role, or perhaps that its loss is compensated by cross talk or redundancy with other pentapeptide repeat proteins.

scholarly journals Septum-Localized Protein Required for Filament Integrity and Diazotrophy in the Heterocyst-Forming Cyanobacterium Anabaena sp. Strain PCC 7120

2007 ◽  
Vol 189 (10) ◽  
pp. 3884-3890 ◽  
Author(s):  
Enrique Flores ◽  
Rafael Pernil ◽  
Alicia M. Muro-Pastor ◽  
Vicente Mariscal ◽  
Iris Maldener ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Nitrogenase Activity ◽  
Early Stage ◽  
Vegetative Cells ◽  
Reading Frame ◽  
Cyanobacterium Anabaena ◽  
In The Wild ◽  
Z Ring ◽  
Green Fluorescent ◽  
Pcc 7120

ABSTRACT Heterocysts, formed when filamentous cyanobacteria, such as Anabaena sp. strain PCC 7120, are grown in the absence of combined nitrogen, are cells that are specialized in fixing atmospheric nitrogen (N2) under oxic conditions and that transfer fixed nitrogen to the vegetative cells of the filament. Anabaena sp. mutants whose sepJ gene (open reading frame alr2338 of the Anabaena sp. genome) was affected showed filament fragmentation and arrested heterocyst differentiation at an early stage. In a sepJ insertional mutant, a layer similar to a heterocyst polysaccharide layer was formed, but the heterocyst-specific glycolipids were not synthesized. The sepJ mutant did not exhibit nitrogenase activity even when assayed under anoxic conditions. In contrast to proheterocysts produced in the wild type, those produced in the sepJ mutant still divided. SepJ is a multidomain protein whose N-terminal region is predicted to be periplasmic and whose C-terminal domain resembles an export permease. Using a green fluorescent protein translationally fused to the carboxyl terminus of SepJ, we observed that in mature heterocysts and vegetative cells, the protein is localized at the intercellular septa, and when cell division starts, it is localized in a ring whose position is similar to that of a Z ring. SepJ is a novel composite protein needed for filament integrity, proper heterocyst development, and diazotrophic growth.

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 Expression and Mutational Analysis of the glnB Genomic Region in the Heterocyst-Forming Cyanobacterium Anabaena sp. Strain PCC 7120

2009 ◽  
Vol 191 (7) ◽  
pp. 2353-2361 ◽  
Author(s):  
Javier Paz-Yepes ◽  
Enrique Flores ◽  
Antonia Herrero
Keyword(s):  
Time Course ◽  
Mutational Analysis ◽  
Nitrogenase Activity ◽  
Genomic Region ◽  
Nitrogen Deprivation ◽  
Transport Activity ◽  
Wild Type ◽  
Combined Nitrogen ◽  
Pcc 7120 ◽  
Diazotrophic Growth

ABSTRACT In the filamentous, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120, the glnB gene is expressed at considerable levels both in the presence and in the absence of combined nitrogen, although induction, influenced by NtcA, takes place upon combined-nitrogen deprivation likely localized to vegetative cells. In spite of extensive efforts, a derivative of PCC 7120 lacking a functional glnB gene could be obtained only with constructs that lead to overexpression of a downstream open reading frames (ORF), particularly all2318. Strain CSP10 [glnB all2318(Con)] exhibited growth rates similar to those of the wild type when it was using nitrate or ammonium, but its diazotrophic growth was impaired. However, it differentiated heterocysts with a time course and distribution pattern similar to those of the wild type, although with no cyanophycin-containing polar granules, and exhibited impaired nitrogenase activity under oxic conditions, but not under microoxic conditions. In the mutant, NtcA-dependent inducion of the hetC and nifH genes was unaltered, but induction of the urtA gene and urea transport activity were increased. Active uptake of nitrite was also increased and insensitive to the ammonium-promoted inhibition observed for the wild type. Thus, regulation of the nitrite transport activity requires the glnB gene product. In the presence of a wild-type glnB gene, neither inactivation nor overexpression of all2318 produced an apparent phenotype. Thus, in an otherwise wild-type background, the glnB gene appears to be essential for growth of strain PCC 7120. For growth with combined nitrogen but not for diazotrophic growth, the requirement for glnB can be overridden by increasing the expression of all2318 (and/or ORFs downstream of it).

scholarly journals Mutations in Four Regulatory Genes Have Interrelated Effects on Heterocyst Maturation in Anabaena sp. Strain PCC 7120

2006 ◽  
Vol 188 (21) ◽  
pp. 7387-7395 ◽  
Author(s):  
Sigal Lechno-Yossef ◽  
Qing Fan ◽  
Shigeki Ehira ◽  
Naoki Sato ◽  
C. Peter Wolk
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Response Regulator ◽  
Regulatory System ◽  
Transcript Abundance ◽  
Regulatory Genes ◽  
Wild Type ◽  
Serine Threonine Kinase ◽  
In The Wild ◽  
Pcc 7120

ABSTRACT Regulatory genes hepK, hepN, henR, and hepS are required for heterocyst maturation in Anabaena sp. strain PCC 7120. They presumptively encode two histidine kinases, a response regulator, and a serine/threonine kinase, respectively. To identify relationships between those genes, we compared global patterns of gene expression, at 14 h after nitrogen step-down, in corresponding mutants and in the wild-type strain. Heterocyst envelopes of mutants affected in any of those genes lack a homogeneous, polysaccharide layer. Those of a henR mutant also lack a glycolipid layer. patA, which encodes a positive effector of heterocyst differentiation, was up-regulated in all mutants except the hepK mutant, suggesting that patA expression may be inhibited by products related to heterocyst development. hepS and hepK were up-regulated if mutated and so appear to be negatively autoregulated. HepS and HenR regulated a common set of genes and so appear to belong to one regulatory system. Some nontranscriptional mechanism may account for the observation that henR mutants lack, and hepS mutants possess, a glycolipid layer, even though both mutations down-regulated genes involved in formation of the glycolipid layer. HepK and HepN also affected transcription of a common set of genes and therefore appear to share a regulatory pathway. However, the transcript abundance of other genes differed very significantly from expression in the wild-type strain in either the hepK or hepN mutant while differing very little from wild-type expression in the other of those two mutants. Therefore, hepK and hepN appear to participate also in separate pathways.

scholarly journals The hetF Gene Product Is Essential to Heterocyst Differentiation and Affects HetR Function in the Cyanobacterium Nostoc punctiforme

2001 ◽  
Vol 183 (8) ◽  
pp. 2654-2661 ◽  
Author(s):  
Francis C. Y. Wong ◽  
John C. Meeks
Keyword(s):  
Fluorescent Protein ◽  
Filamentous Cyanobacterium ◽  
Multicopy Plasmid ◽  
Wild Type ◽  
Nostoc Punctiforme ◽  
Combined Nitrogen ◽  
In The Wild ◽  
Step Down ◽  
Green Fluorescent ◽  
Heterocyst Development

ABSTRACT A novel gene, hetF, was identified as essential for heterocyst development in the filamentous cyanobacterium Nostoc punctiforme strain ATCC 29133. In the absence of combined nitrogen, hetF mutants were unable to differentiate heterocysts, whereas extra copies of hetF intrans induced the formation of clusters of heterocysts. Sequences hybridizing to a hetF probe were detected only in heterocyst-forming cyanobacteria. The inactivation and multicopy effects of hetF were similar to those of hetR, which encodes a self-degrading serine protease thought to be a central regulator of heterocyst development. Increased transcription ofhetR begins in developing cells 3 to 6 h after deprivation for combined nitrogen (N step-down), and the HetR protein specifically accumulates in heterocysts. In the hetFmutant, this increase in hetR transcription was delayed, and a hetR promoter::green fluorescent protein (GFP) transcriptional reporter indicated that increased transcription of hetR occurred in all cells rather than only in developing heterocysts. When a fully functional HetR-GFP fusion protein was expressed in the hetF mutant from a multicopy plasmid, HetR-GFP accumulated nonspecifically in all cells under nitrogen-replete conditions; when expressed in the wild type, HetR-GFP was observed only in heterocysts after N step-down. HetF therefore appears to cooperate with HetR in a positive regulatory pathway and may be required for the increased transcription of hetR and localization of the HetR protein in differentiating heterocysts.

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.

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