Cold-stress-altered phosphorylation of EF-Tu in the cyanobacterium Anabaena sp. strain PCC 7120

2007 ◽  
Vol 53 (5) ◽  
pp. 551-558 ◽  
Author(s):  
Bassam El-Fahmawi ◽  
George W. Owttrim
Keyword(s):  
Cold Stress ◽  
Prolonged Exposure ◽  
De Novo ◽  
Affinity Purification ◽  
Translation Elongation ◽  
Cyanobacterium Anabaena ◽  
Anabaena Sp ◽  
Pcc 7120

Growth of prokaryotes at reduced temperature results in the formation of a cold-adapted ribosome through association with de novo synthesized polypeptides. In vitro and in vivo phosphorylation studies combined with affinity purification and mass spectrometry identified that the phosphorylation status of translation elongation factor EF-Tu was altered in response to cold stress in the photosynthetic, Gram-negative cyanobacterium Anabaena sp. strain PCC 7120. In response to a temperature downshift from 30 to 20 °C, EF-Tu was rapidly and transiently hyperphosphorylated during the acclimation phase followed by a reduction in phosphorylation below background levels in response to prolonged exposure. EF-Tu was identified as a phosphothreonine protein. Unexpectedly, ribosomal protein S2 was also observed to be a phosphoprotein continuously phosphorylated during cold stress. The phosphorylation status of EF-Tu has previously been associated with translational regulation in other systems, with a reduction in translation elongation occurring in response to phosphorylation. These results provide evidence for a novel mechanism by which translation is initially downregulated in response to cold stress in Anabaena.

scholarly journals Biosensors-Based In Vivo Quantification of 2-Oxoglutarate in Cyanobacteria and Proteobacteria

Life ◽  
2018 ◽  
Vol 8 (4) ◽  
pp. 51 ◽  
Author(s):  
Hai-Lin Chen ◽  
Amel Latifi ◽  
Cheng-Cai Zhang ◽  
Christophe Bernard
Keyword(s):  
Conformational Changes ◽  
Nitrogen Assimilation ◽  
Krebs Cycle ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cellular Level ◽  
Cyanobacterium Anabaena ◽  
Anabaena Sp ◽  
Pcc 7120

2-oxoglutarate (α-ketoglutarate; 2-OG) is an intermediate of the Krebs cycle, and constitutes the carbon skeleton for nitrogen assimilation and the synthesis of a variety of compounds. In addition to being an important metabolite, 2-OG is a signaling molecule with a broad regulatory repertoire in a variety of organisms, including plants, animals, and bacteria. Although challenging, measuring the levels and variations of metabolic signals in vivo is critical to better understand how cells control specific processes. To measure cellular 2-OG concentrations and dynamics, we designed a set of biosensors based on the fluorescence resonance energy transfer (FRET) technology that can be used in vivo in different organisms. For this purpose, we took advantage of the conformational changes of two cyanobacterial proteins induced by 2-OG binding. We show that these biosensors responded immediately and specifically to different 2-OG levels, and hence allowed to measure 2-OG variations in function of environmental modifications in the proteobacterium Escherichia coli and in the cyanobacterium Anabaena sp. PCC 7120. Our results pave the way to study 2-OG dynamics at the cellular level in uni- and multi-cellular organisms.

scholarly journals NrrA Directly Regulates Expression of hetR during Heterocyst Differentiation in the Cyanobacterium Anabaena sp. Strain PCC 7120

2006 ◽  
Vol 188 (24) ◽  
pp. 8520-8525 ◽  
Author(s):  
Shigeki Ehira ◽  
Masayuki Ohmori
Keyword(s):  
Transcription Initiation ◽  
Response Regulator ◽  
Nitrogen Deprivation ◽  
Heterocyst Differentiation ◽  
Combined Nitrogen ◽  
Regulatory Cascade ◽  
Cyanobacterium Anabaena ◽  
Anabaena Sp ◽  
Pcc 7120

ABSTRACT Heterocyst differentiation in the cyanobacterium Anabaena sp. strain PCC 7120 requires NtcA, the global nitrogen regulator in cyanobacteria, and HetR, the master regulator of heterocyst differentiation. Expression of hetR is upregulated by nitrogen deprivation, and its upregulation depends on NtcA. However, it has not yet been revealed how NtcA regulates the expression of hetR. In the experiments presented here, it was confirmed that NrrA (All4312), a nitrogen-responsive response regulator, was required for the upregulation of hetR. The use of the nitrogen-responsive transcription initiation sites (TISs) for the hetR gene depended upon NrrA. NrrA bound specifically to the region upstream of TISs located at positions −728 and −696 in vitro. Overexpression of nrrA resulted in enhanced hetR expression and heterocyst formation. A molecular regulatory cascade is proposed whereby NtcA upregulates the expression of nrrA upon limitation of combined nitrogen in the medium and then NrrA upregulates the expression of hetR, leading to heterocyst differentiation.

scholarly journals Thioredoxin Dependent Changes in the Redox States of FurA from Anabaena sp. PCC 7120

Antioxidants ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 913
Author(s):  
Jorge Guío ◽  
María Teresa Bes ◽  
Mónica Balsera ◽  
Laura Calvo-Begueria ◽  
Emma Sevilla ◽  
...  
Keyword(s):  
Reductase Activity ◽  
Thioredoxin Reductase ◽  
Zinc Ion ◽  
Exchange Reactions ◽  
Redox Partner ◽  
Redox Partners ◽  
Anabaena Sp ◽  
Pcc 7120

FurA is a multifunctional regulator in cyanobacteria that contains five cysteines, four of them arranged into two CXXC motifs. Lack of a structural zinc ion enables FurA to develop disulfide reductase activity. In vivo, FurA displays several redox isoforms, and the oxidation state of its cysteines determines its activity as regulator and its ability to bind different metabolites. Because of the relationship between FurA and the control of genes involved in oxidative stress defense and photosynthetic metabolism, we sought to investigate the role of type m thioredoxin TrxA as a potential redox partner mediating dithiol-disulfide exchange reactions necessary to facilitate the interaction of FurA with its different ligands. Both in vitro cross-linking assays and in vivo two-hybrid studies confirmed the interaction between FurA and TrxA. Light to dark transitions resulted in reversible oxidation of a fraction of the regulator present in Anabaena sp. PCC7120. Reconstitution of an electron transport chain using E. coli NADPH-thioredoxin-reductase followed by alkylation of FurA reduced cysteines evidenced the ability of TrxA to reduce FurA. Furthermore, the use of site-directed mutants allowed us to propose a plausible mechanism for FurA reduction. These results point to TrxA as one of the redox partners that modulates FurA performance.

scholarly journals ThetrpEGene Negatively Regulates Differentiation of Heterocysts at the Level of Induction in Anabaena sp. Strain PCC 7120

2014 ◽  
Vol 197 (2) ◽  
pp. 362-370 ◽  
Author(s):  
Patrick Videau ◽  
Loralyn M. Cozy ◽  
Jasmine E. Young ◽  
Blake Ushijima ◽  
Reid T. Oshiro ◽  
...  
Keyword(s):  
Repeat Sequence ◽  
Mutant Phenotype ◽  
Wild Type ◽  
Content Type ◽  
Type Pattern ◽  
In The Wild ◽  
Anabaena Sp ◽  
Pcc 7120

Levels of 2-oxoglutarate (2-OG) reflect nitrogen status in many bacteria. In heterocystous cyanobacteria, a spike in the 2-OG level occurs shortly after the removal of combined nitrogen from cultures and is an integral part of the induction of heterocyst differentiation. In this work, deletion of one of the two annotatedtrpEgenes inAnabaenasp. strain PCC 7120 resulted in a spike in the 2-OG level and subsequent differentiation of a wild-type pattern of heterocysts when filaments of the mutant were transferred from growth on ammonia to growth on nitrate. In contrast, 2-OG levels were unaffected in the wild type, which did not differentiate under the same conditions. An inverted-repeat sequence located upstream oftrpEbound a central regulator of differentiation, HetR,in vitroand was necessary for HetR-dependent transcription of a reporter fusion and complementation of the mutant phenotypein vivo. Functional complementation of the mutant phenotype with the addition of tryptophan suggested that levels of tryptophan, rather than the demonstrated anthranilate synthase activity of TrpE, mediated the developmental response of the wild type to nitrate. A model is presented for the observed increase in 2-OG in thetrpEmutant.

scholarly journals Glycolipid composition of the heterocyst envelope of Anabaena sp. PCC 7120 is crucial for diazotrophic growth and relies on the UDP-galactose 4-epimerase HgdA

2018 ◽  
Author(s):  
Dmitry Shvarev ◽  
Carolina N. Nishi ◽  
Iris Maldener
Keyword(s):  
Cell Envelope ◽  
Functional Characterization ◽  
Site Directed Mutagenesis ◽  
Transmission Electron ◽  
Complex Process ◽  
Cyanobacterium Anabaena ◽  
Anabaena Sp ◽  
Pcc 7120 ◽  
Diazotrophic Growth

The nitrogenase complex in the heterocysts of the filamentous freshwater cyanobacterium Anabaena sp. PCC 7120 fixes atmospheric nitrogen to allow diazotrophic growth. The heterocyst cell envelope protects the nitrogenase from oxygen and consists of a polysaccharide and a glycolipid layer that are formed by a complex process involving the recruitment of different proteins. Here we studied the function of the putative nucleoside-diphosphate-sugar epimerase HgdA, which along with HgdB and HgdC is essential for deposition of the glycolipid layer and growth without a combined nitrogen source. Using site-directed mutagenesis and single homologous recombination approach, we performed a thoroughly functional characterization of HgdA and confirmed that the glycolipid layer of the hgdA mutant heterocyst is aberrant as shown by transmission electron microscopy and chemical analysis. The hgdA gene was expressed during late stages of the heterocyst differentiation. GFP-tagged HgdA protein localized inside the heterocysts. The purified HgdA protein had UDP-galactose 4-epimerase activity in vitro. This enzyme could be responsible for synthesis of heterocyst-specific glycolipid precursors, which could be transported over the cell wall by the ABC transporter components HgdB/HgdC.

scholarly journals Role of Two NtcA-Binding Sites in the Complex ntcA Gene Promoter of the Heterocyst-Forming Cyanobacterium Anabaena sp. Strain PCC 7120

2008 ◽  
Vol 190 (22) ◽  
pp. 7584-7590 ◽  
Author(s):  
Elvira Olmedo-Verd ◽  
Ana Valladares ◽  
Enrique Flores ◽  
Antonia Herrero ◽  
Alicia M. Muro-Pastor
Keyword(s):  
Binding Sites ◽  
Gene Promoter ◽  
Filamentous Cyanobacterium ◽  
Mobility Shift ◽  
Dnase I Footprinting ◽  
Cyanobacterium Anabaena ◽  
Anabaena Sp ◽  
Pcc 7120

ABSTRACT Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that fixes N2 in specialized cells called heterocysts, which differentiate from vegetative cells in a process that requires the nitrogen control transcription factor NtcA. 2-Oxoglutarate-stimulated binding of purified NtcA to wild-type and modified versions of the ntcA gene promoter from Anabaena sp. was analyzed by mobility shift and DNase I footprinting assays, and the role of NtcA-binding sites in the expression of the ntcA gene during heterocyst differentiation was studied in vivo by using an ntcA-gfp translational fusion and primer extension analysis. Mutation of neither of the two identified NtcA-binding sites eliminated localized expression of ntcA in proheterocysts, but mutation of both sites led to very low, nonlocalized expression.

Expanding the Natural Products Heterologous Expression Repertoire in the Model Cyanobacterium Anabaena sp. Strain PCC 7120: Production of Pendolmycin and Teleocidin B-4

2019 ◽  
Author(s):  
Patrick Videau ◽  
Kaitlyn Wells ◽  
Arun Singh ◽  
Jessie Eiting ◽  
Philip Proteau ◽  
...  
Keyword(s):  
Natural Products ◽  
Genome Mining ◽  
Gene Clusters ◽  
Combinatorial Biosynthesis ◽  
Test Case ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Cyanobacterium Anabaena ◽  
Anabaena Sp ◽  
Pcc 7120

Cyanobacteria are prolific producers of natural products and genome mining has shown that many orphan biosynthetic gene clusters can be found in sequenced cyanobacterial genomes. New tools and methodologies are required to investigate these biosynthetic gene clusters and here we present the use of <i>Anabaena </i>sp. strain PCC 7120 as a host for combinatorial biosynthesis of natural products using the indolactam natural products (lyngbyatoxin A, pendolmycin, and teleocidin B-4) as a test case. We were able to successfully produce all three compounds using codon optimized genes from Actinobacteria. We also introduce a new plasmid backbone based on the native <i>Anabaena</i>7120 plasmid pCC7120ζ and show that production of teleocidin B-4 can be accomplished using a two-plasmid system, which can be introduced by co-conjugation.

Target Gene Inactivation in Cyanobacterium Anabaena sp. PCC 7120

BIO-PROTOCOL ◽  
2016 ◽  
Vol 6 (15) ◽  
Author(s):  
Kangming Chen ◽  
Huilan Zhu ◽  
Liping Gu ◽  
Shengni Tian ◽  
Ruanbao Zhou
Keyword(s):  
Target Gene ◽  
Gene Inactivation ◽  
Cyanobacterium Anabaena ◽  
Anabaena Sp ◽  
Pcc 7120

scholarly journals USP9X-mediated KDM4C deubiquitination promotes lung cancer radioresistance by epigenetically inducing TGF-β2 transcription

2021 ◽  
Author(s):  
Xiaohua Jie ◽  
William Pat Fong ◽  
Rui Zhou ◽  
Ye Zhao ◽  
Yingchao Zhao ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Affinity Purification ◽  
Smad Signaling ◽  
Lung Cancer Patients ◽  
Lysine Specific Demethylase ◽  
Underlying Mechanisms ◽  
H3k9me3 Level ◽  
Critical Binding

AbstractRadioresistance is regarded as the main barrier to effective radiotherapy in lung cancer. However, the underlying mechanisms of radioresistance remain elusive. Here, we show that lysine-specific demethylase 4C (KDM4C) is overexpressed and correlated with poor prognosis in lung cancer patients. We provide evidence that genetical or pharmacological inhibition of KDM4C impairs tumorigenesis and radioresistance in lung cancer in vitro and in vivo. Moreover, we uncover that KDM4C upregulates TGF-β2 expression by directly reducing H3K9me3 level at the TGF-β2 promoter and then activates Smad/ATM/Chk2 signaling to confer radioresistance in lung cancer. Using tandem affinity purification technology, we further identify deubiquitinase USP9X as a critical binding partner that deubiquitinates and stabilizes KDM4C. More importantly, depletion of USP9X impairs TGF-β2/Smad signaling and radioresistance by destabilizing KDM4C in lung cancer cells. Thus, our findings demonstrate that USP9X-mediated KDM4C deubiquitination activates TGF-β2/Smad signaling to promote radioresistance, suggesting that targeting KDM4C may be a promising radiosensitization strategy in the treatment of lung cancer.

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