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 Catabolic Function of Compartmentalized Alanine Dehydrogenase in the Heterocyst-Forming Cyanobacterium Anabaena sp. Strain PCC 7120

2010 ◽  
Vol 192 (19) ◽  
pp. 5165-5172 ◽  
Author(s):  
Rafael Pernil ◽  
Antonia Herrero ◽  
Enrique Flores
Keyword(s):  
Fluorescent Protein ◽  
Nitrogenase Activity ◽  
Vegetative Cells ◽  
Reading Frame ◽  
Alanine Dehydrogenase ◽  
Cyanobacterium Anabaena ◽  
Green Fluorescent ◽  
Pcc 7120 ◽  
Diazotrophic Growth

ABSTRACT In the diazotrophic filaments of heterocyst-forming cyanobacteria, an exchange of metabolites takes place between vegetative cells and heterocysts that results in a net transfer of reduced carbon to the heterocysts and of fixed nitrogen to the vegetative cells. Open reading frame alr2355 of the genome of Anabaena sp. strain PCC 7120 is the ald gene encoding alanine dehydrogenase. A strain carrying a green fluorescent protein (GFP) fusion to the N terminus of Ald (Ald-N-GFP) showed that the ald gene is expressed in differentiating and mature heterocysts. Inactivation of ald resulted in a lack of alanine dehydrogenase activity, a substantially decreased nitrogenase activity, and a 50% reduction in the rate of diazotrophic growth. Whereas production of alanine was not affected in the ald mutant, in vivo labeling with [14C]alanine (in whole filaments and isolated heterocysts) or [14C]pyruvate (in whole filaments) showed that alanine catabolism was hampered. Thus, alanine catabolism in the heterocysts is needed for normal diazotrophic growth. Our results extend the significance of a previous work that suggested that alanine is transported from vegetative cells into heterocysts in the diazotrophic Anabaena filament.

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 Mutation in yaaT Leads to Significant Inhibition of Phosphorelay during Sporulation in Bacillus subtilis

2002 ◽  
Vol 184 (20) ◽  
pp. 5545-5553 ◽  
Author(s):  
Shigeo Hosoya ◽  
Kei Asai ◽  
Naotake Ogasawara ◽  
Michio Takeuchi ◽  
Tsutomu Sato
Keyword(s):  
Bacillus Subtilis ◽  
Fluorescent Protein ◽  
Early Stage ◽  
Response Regulator ◽  
Significant Inhibition ◽  
Open Reading Frame ◽  
Histidine Kinases ◽  
Reading Frame ◽  
Green Fluorescent ◽  
Sporulation Genes

ABSTRACT In the course of a Bacillus subtilis functional genomics project which involved screening for sporulation genes, we identified an open reading frame, yaaT, whose disruptant exhibits a sporulation defect. Twenty-four hours after the initiation of sporulation, most cells of the yaaT mutant exhibited stage 0 of sporulation, indicating that the yaaT mutation blocks sporulation at an early stage. Furthermore, the mutation in yaaT led to a significant decrease in transcription from a promoter controlled by Spo0A, a key response regulator required for the initiation of sporulation. However, neither the level of transcription of spo0A, the activity of σH, which transcribes spo0A, nor the amount of Spo0A protein was severely affected by the mutation in yaaT. Bypassing the phosphorelay by introducing an spo0A mutation (sof-1) into the yaaT mutant suppressed the sporulation defect, suggesting that the yaaT mutation interferes with the phosphorelay process comprising Spo0F, Spo0B, and histidine kinases. We also observed that mutation of spo0E, which encodes the phosphatase that dephosphorylates Spo0A-P, suppressed the sporulation defect in the yaaT mutant. These results strongly suggest that yaaT plays a significant role in the transduction of signals to the phosphorelay for initiation of sporulation. Micrographs indicated that YaaT-green fluorescent protein localizes to the peripheral membrane, as well as to the septum, during sporulation.

scholarly journals Posttranscriptional Regulation of Glutamine Synthetase in the Filamentous Cyanobacterium Anabaena sp. PCC 7120: Differential Expression between Vegetative Cells and Heterocysts

2010 ◽  
Vol 192 (18) ◽  
pp. 4701-4711 ◽  
Author(s):  
Carla V. Galmozzi ◽  
Lorena Saelices ◽  
Francisco J. Florencio ◽  
M. Isabel Muro-Pastor
Keyword(s):  
Glutamine Synthetase ◽  
Differential Expression ◽  
Posttranscriptional Regulation ◽  
Filamentous Cyanobacterium ◽  
Vegetative Cells ◽  
Amino Terminal ◽  
Cyanobacterium Anabaena ◽  
In The Wild ◽  
Pcc 7120

ABSTRACT Genes homologous to those implicated in glutamine synthetase (GS) regulation by protein-protein interaction in the cyanobacterium Synechocystis sp. strain PCC 6803 are conserved in several cyanobacterial sequenced genomes. We investigated this GS regulatory mechanism in Anabaena sp. strain PCC 7120. In this strain the system operates with only one GS inactivation factor (inactivation factor 7A [IF7A]), encoded by open reading frame (ORF) asl2329 (gifA). Following addition of ammonium, expression of gifA is derepressed, leading to the synthesis of IF7A, and consequently, GS is inactivated. Upon ammonium removal, the GS activity returns to the initial level and IF7A becomes undetectable. The global nitrogen control protein NtcA binds to the gifA promoter. Constitutive high expression levels of gifA were found in an Anabaena ntcA mutant (CSE2), indicating a repressor role for NtcA. In vitro studies demonstrate that Anabaena GS is not inactivated by Synechocystis IFs (IF7 and IF17), indicating the specificity of the system. We constructed an Anabaena strain expressing a second inactivating factor, containing the amino-terminal part of IF17 from Synechocystis fused to IF7A. GS inactivation in this strain is more effective than that in the wild type (WT) and resembles that observed in Synechocystis. Finally we found differential expression of the IF system between heterocysts and vegetative cells of Anabaena.

scholarly journals Newly Identified Cytochrome c Oxidase Operon in the Nitrogen-Fixing Cyanobacterium Anabaena sp. Strain PCC 7120 Specifically Induced in Heterocysts

2002 ◽  
Vol 184 (9) ◽  
pp. 2491-2499 ◽  
Author(s):  
Kathryn M. Jones ◽  
Robert Haselkorn
Keyword(s):  
Fluorescent Protein ◽  
Low Oxygen ◽  
Nucleotide Level ◽  
Gene Encoding ◽  
Terminal Oxidases ◽  
Cyanobacterium Anabaena ◽  
Anabaena Sp ◽  
Green Fluorescent ◽  
Pcc 7120 ◽  
Late Stages

ABSTRACT Two operons have been cloned from Anabaena sp. strain PCC 7120 DNA, each of which encodes the three core subunits of distinct mitochondrial-type cytochrome c oxidases. The two operons are only 72 to 85% similar to one another at the nucleotide level in the most conserved subunit. One of these, coxBACII, is induced >20-fold in the middle to late stages of heterocyst differentiation. Analysis of green fluorescent protein reporters indicates that this operon is expressed specifically in proheterocysts and heterocysts. The other operon, coxBACI, is induced only 2.5-fold following nitrogen step-down and is expressed in all cells. Surprisingly, a disruption mutant of coxAII, the gene encoding subunit I of the heterocyst-specific oxidase, grows normally in the absence of combined nitrogen. It is likely that coxBACI and/or two other putative terminal oxidases present in the Anabaena sp. strain PCC 7120 genome are able to compensate for the loss of the heterocyst-specific oxidase in providing ATP for nitrogen fixation and maintaining a low oxygen level in heterocysts.

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 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 An amidase is required for proper intercellular communication in the filamentous cyanobacteriumAnabaenasp. PCC 7120

2017 ◽  
Vol 114 (8) ◽  
pp. E1405-E1412 ◽  
Author(s):  
Zhenggao Zheng ◽  
Amin Omairi-Nasser ◽  
Xiying Li ◽  
Chunxia Dong ◽  
Yan Lin ◽  
...  
Keyword(s):  
Intercellular Communication ◽  
Electron Tomography ◽  
Filamentous Cyanobacterium ◽  
Channel Formation ◽  
Wild Type ◽  
In The Wild ◽  
Z Ring ◽  
Green Fluorescent ◽  
20 Nm ◽  
Pcc 7120

Channels that cross cell walls and connect the cytoplasm of neighboring cells in multicellular cyanobacteria are pivotal for intercellular communication. We find that the product of the geneall1140of the filamentous cyanobacteriumAnabaenasp. PCC 7120 is required for proper channel formation.All1140encodes an amidase that hydrolyses purified peptidoglycans. An All1140-GFP fusion protein is located at the Z-ring in the periplasmic space during most of the cell cycle. Anall1140-null mutant (M40) was unable to grow diazotrophically, and no mature heterocysts were observed in the absence of combined nitrogen. Expression of two key genes,hetRandpatS, was studied in M40 using GFP as a reporter. Upon nitrogen step-down, the patterned distribution of green fluorescent cells in filaments seen in the wild type were not observed in mutant M40. Intercellular communication in M40 was studied by measuring fluorescence recovery after photobleaching (FRAP). Movement of calcein (622 Da) was aborted in M40, suggesting that the channels connecting the cytoplasm of neighboring cells are impaired in the mutant. The channels were examined with electron tomography; their diameters were nearly identical, 12.7 nm for the wild type and 12.4 nm for M40, suggesting that AmiC3 is not required for channel formation. However, when the cell wall sacculi isolated by boiling were examined by EM, the average sizes of the channels of the wild type and M40 were 20 nm and 12 nm, respectively, suggesting that the channel walls of the wild type are expandable and that this expandability requires AmiC3.

scholarly journals Two Genes Encoding Protein Kinases of the HstK Family Are Involved in Synthesis of the Minor Heterocyst-Specific Glycolipid in the Cyanobacterium Anabaena sp. Strain PCC 7120

2007 ◽  
Vol 189 (14) ◽  
pp. 5075-5081 ◽  
Author(s):  
Lei Shi ◽  
Jian-Hong Li ◽  
Yong Cheng ◽  
Li Wang ◽  
Wen-Li Chen ◽  
...  
Keyword(s):  
Nitrogenase Activity ◽  
Early Stage ◽  
Kinase Domain ◽  
Filamentous Cyanobacterium ◽  
Regulatory Pathways ◽  
Genes Encoding ◽  
Putative Transcription Factor ◽  
Oxygen Sensitive ◽  
Cyanobacterium Anabaena ◽  
Pcc 7120

ABSTRACT The filamentous cyanobacterium Anabaena sp. strain PCC 7120 can fix N2 under oxic conditions, and the activity of nitrogen fixation occurs exclusively in heterocysts, cells differentiated from vegetative cells in response to a limitation of a combined-nitrogen source in the growth medium. At the late stages of heterocyst differentiation, an envelope layer composed of two glycolipids is formed to limit the entry of oxygen so that the oxygen-sensitive nitrogenase can function. The genome of Anabaena sp. strain PCC 7120 possesses a family of 13 genes (the hstK family), all encoding proteins with a putative Ser/Thr kinase domain at their N termini and a His-kinase domain at their C termini. In this study, we showed that the double mutant D4.3 strain, in which two members of this gene family, pkn44 (all1625) and pkn30 (all3691), were both inactivated, failed to fix N2 in the presence of oxygen (Fox−). In an environment without oxygen, a low level of nitrogenase activity was detectable (Fix+). Heterocyst development in the mutant D4.3 was delayed by 24 h and arrested at a relatively early stage without the formation of the glycolipid layer (Hgl−). Only the minor species of the two heterocyst-specific glycolipids (HGLs) was missing in the mutant. We propose that DevH, a putative transcription factor, coordinates the synthesis of both HGLs, while Pkn44/Pkn30 and the previously characterized PrpJ may represent two distinct regulatory pathways involved in the synthesis of the minor HGL and the major HGL, respectively.

scholarly journals Sigma Factor Genes sigC, sigE, and sigG Are Upregulated in Heterocysts of the Cyanobacterium Anabaena sp. Strain PCC 7120

2007 ◽  
Vol 189 (22) ◽  
pp. 8392-8396 ◽  
Author(s):  
M. Ramona Aldea ◽  
Rodrigo A. Mella-Herrera ◽  
James W. Golden
Keyword(s):  
Sigma Factor ◽  
Fluorescent Protein ◽  
Developmental Regulation ◽  
Time Lapse ◽  
Green Fluorescent Protein Fluorescence ◽  
Protein Fluorescence ◽  
Cyanobacterium Anabaena ◽  
Reporter Strains ◽  
Green Fluorescent ◽  
Pcc 7120

ABSTRACT We used gfp transcriptional fusions to investigate the regulation of eight sigma factor genes during heterocyst development in the cyanobacterium Anabaena sp. strain PCC 7120. Reporter strains containing gfp fusions with the upstream regions of sigB2, sigD, sigI, and sigJ did not show developmental regulation. Time-lapse microscopy of sigC, sigE, and sigG reporter strains showed increased green fluorescent protein fluorescence in differentiating cells at 4 h, 16 h, and 9 h, respectively, after nitrogen step down.

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