scholarly journals Synechocystis KaiC3 Displays Temperature- and KaiB-Dependent ATPase Activity and Is Important for Growth in Darkness

2019 ◽  
Vol 202 (4) ◽  
Author(s):  
Anika Wiegard ◽  
Christin Köbler ◽  
Katsuaki Oyama ◽  
Anja K. Dörrich ◽  
Chihiro Azai ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Circadian Clock ◽  
Atp Hydrolysis ◽  
Synechococcus Elongatus ◽  
Constant Darkness ◽  
Content Type ◽  
Clock Proteins ◽  
Pcc 7942 ◽  
Pcc 6803

ABSTRACT Cyanobacteria form a heterogeneous bacterial group with diverse lifestyles, acclimation strategies, and differences in the presence of circadian clock proteins. In Synechococcus elongatus PCC 7942, a unique posttranslational KaiABC oscillator drives circadian rhythms. ATPase activity of KaiC correlates with the period of the clock and mediates temperature compensation. Synechocystis sp. strain PCC 6803 expresses additional Kai proteins, of which KaiB3 and KaiC3 proteins were suggested to fine-tune the standard KaiAB1C1 oscillator. In the present study, we therefore characterized the enzymatic activity of KaiC3 as a representative of nonstandard KaiC homologs in vitro. KaiC3 displayed ATPase activity lower than that of the Synechococcus elongatus PCC 7942 KaiC protein. ATP hydrolysis was temperature dependent. Hence, KaiC3 is missing a defining feature of the model cyanobacterial circadian oscillator. Yeast two-hybrid analysis showed that KaiC3 interacts with KaiB3, KaiC1, and KaiB1. Further, KaiB3 and KaiB1 reduced in vitro ATP hydrolysis by KaiC3. Spot assays showed that chemoheterotrophic growth in constant darkness is completely abolished after deletion of ΔkaiAB1C1 and reduced in the absence of kaiC3. We therefore suggest a role for adaptation to darkness for KaiC3 as well as a cross talk between the KaiC1- and KaiC3-based systems. IMPORTANCE The circadian clock influences the cyanobacterial metabolism, and deeper understanding of its regulation will be important for metabolic optimizations in the context of industrial applications. Due to the heterogeneity of cyanobacteria, characterization of clock systems in organisms apart from the circadian model Synechococcus elongatus PCC 7942 is required. Synechocystis sp. strain PCC 6803 represents a major cyanobacterial model organism and harbors phylogenetically diverged homologs of the clock proteins, which are present in various other noncyanobacterial prokaryotes. By our in vitro studies we unravel the interplay of the multiple Synechocystis Kai proteins and characterize enzymatic activities of the nonstandard clock homolog KaiC3. We show that the deletion of kaiC3 affects growth in constant darkness, suggesting its involvement in the regulation of nonphotosynthetic metabolic pathways.

scholarly journals Synechocystis KaiC3 displays temperature and KaiB dependent ATPase activity and is important for growth in darkness

2019 ◽  
Author(s):  
Anika Wiegard ◽  
Christin Kobler ◽  
Katsuaki Oyama ◽  
Anja Katharina Dorrich ◽  
Chihiro Azai ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Synechococcus Elongatus ◽  
Constant Darkness ◽  
Temperature Dependent ◽  
Clock Proteins ◽  
Circadian Clock Proteins ◽  
Pcc 7942 ◽  
Fine Tune

Cyanobacteria form a heterogeneous bacterial group with diverse lifestyles, acclimation strategies and differences in the presence of circadian clock proteins. In Synechococcus elongatus PCC 7942, a unique posttranslational KaiABC oscillator drives circadian rhythms. ATPase activity of KaiC correlates with the period of the clock and mediates temperature compensation. Synechocystis sp. PCC 6803 expresses additional Kai proteins, of which KaiB3 and KaiC3 proteins were suggested to fine-tune the standard KaiAB1C1 oscillator. In the present study, we therefore characterized the enzymatic activity of KaiC3 as a representative of non-standard KaiC homologs in vitro. KaiC3 displayed ATPase activity, which were lower compared to the Synechococcus elongatus PCC 7942 KaiC protein. ATP hydrolysis was temperature-dependent. Hence, KaiC3 is missing a defining feature of the model cyanobacterial circadian oscillator. Yeast two-hybrid analysis showed that KaiC3 interacts with KaiB3, KaiC1 and KaiB1. Further, KaiB3 and KaiB1 reduced in vitro ATP hydrolysis by KaiC3. Spot assays showed that chemoheterotrophic growth in constant darkness is completely abolished after deletion of ΔkaiAB1C1 and reduced in the absence of kaiC3. We therefore suggest a role for adaptation to darkness for KaiC3 as well as a crosstalk between the KaiC1 and KaiC3 based systems.

scholarly journals Single-molecular and Ensemble-level Oscillations of Cyanobacterial Circadian Clock

2018 ◽  
Author(s):  
Sumita Das ◽  
Tomoki P. Terada ◽  
Masaki Sasai
Keyword(s):  
Atpase Activity ◽  
Circadian Clock ◽  
Single Molecule ◽  
Structural Changes ◽  
Atp Hydrolysis ◽  
Theoretical Models ◽  
Oscillation Period ◽  
Coarse Grained ◽  
Simulation Results

AbstractWhen three cyanobacterial proteins, KaiA, KaiB, and KaiC, are incubated with ATP in vitro, the phosphorylation level of KaiC hexamers shows stable oscillation with approximately 24 h period. In order to understand this KaiABC clockwork, we need to analyze both the macroscopic synchronization of a large number of KaiC hexamers and the microscopic reactions and structural changes in individual KaiC molecules. In the present paper, we explain two coarse-grained theoretical models, the many-molecule (MM) model and the single-molecule (SM) model, to bridge the gap between macroscopic and microscopic understandings. In the simulation results with these models, ATP hydrolysis drives oscillation of individual KaiC hexamers and ATP hydrolysis is necessary for synchronizing oscillations of a large number of KaiC hexamers. Sensitive temperature dependence of the lifetime of the ADP bound state in the CI domain of KaiC hexamers makes the oscillation period temperature insensitive. ATPase activity is correlated to the frequency of phosphorylation oscillation in the single molecule of KaiC hexamer, which should be the origin of the observed ensemble-level correlation between the ATPase activity and the frequency of phosphorylation oscillation. Thus, the simulation results with the MM and SM models suggest that ATP hydrolysis randomly occurring in each CI domain of individual KaiC hexamers is a key process for oscillatory behaviors of the ensemble of many KaiC hexamers.Significance StatementCyanobacterial proteins, KaiA, KaiB, and KaiC, can reconstitute a circadian clock when they are incubated with ATP in vitro. In order to understand this prototypical oscillator, we need to analyze both synchronization of a macroscopically large number of oscillating molecules and microscopic reactions in individual molecules. We introduced two theoretical models to unify macroscopic and microscopic viewpoints. Simulation results suggest that ATP hydrolysis is necessary for synchronization and temperature compensation and that ATPase activity is correlated to the oscillation frequency in individual molecules. Thus, ATP hydrolysis randomly occurring in individual molecules should determine important features of the ensemble-level oscillation.

scholarly journals The Putative Eukaryote-LikeO-GlcNAc Transferase of the Cyanobacterium Synechococcus elongatus PCC 7942 Hydrolyzes UDP-GlcNAc and Is Involved in Multiple Cellular Processes

2014 ◽  
Vol 197 (2) ◽  
pp. 354-361 ◽  
Author(s):  
Kerry A. Sokol ◽  
Neil E. Olszewski
Keyword(s):  
Deletion Mutant ◽  
Bacterial Species ◽  
Synechococcus Elongatus ◽  
Single Amino Acid ◽  
Content Type ◽  
Cellular Processes ◽  
Mutant Phenotypes ◽  
Glcnac Transferase ◽  
Pcc 7942 ◽  
Mutant Cells

The posttranslational addition of a single O-linked β-N-acetylglucosamine (O-GlcNAc) to serine or threonine residues regulates numerous metazoan cellular processes. The enzyme responsible for this modification,O-GlcNAc transferase (OGT), is conserved among a wide variety of organisms and is critical for the viability of many eukaryotes. Although OGTs with domain structures similar to those of eukaryotic OGTs are predicted for many bacterial species, the cellular roles of these OGTs are unknown. We have identified a putative OGT in the cyanobacteriumSynechococcus elongatusPCC 7942 that shows active-site homology and similar domain structure to eukaryotic OGTs. An OGT deletion mutant was created and found to exhibit several phenotypes. Without agitation, mutant cells aggregate and settle out of the medium. The mutant cells have higher free inorganic phosphate levels, wider thylakoid lumen, and differential accumulation of electron-dense inclusion bodies. These phenotypes are rescued by reintroduction of the wild-type OGT but are not fully rescued by OGTs with single amino acid substitutions corresponding to mutations that reduce eukaryotic OGT activity.S. elongatusOGT purified fromEscherichia colihydrolyzed the sugar donor, UDP-GlcNAc, while the mutant OGTs that did not fully rescue the deletion mutant phenotypes had reduced or no activity. These results suggest that bacterial eukaryote-like OGTs, like their eukaryotic counterparts, influence multiple processes.

scholarly journals Photophysiological and Photosynthetic Complex Changes during Iron Starvation in Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942

PLoS ONE ◽  
2013 ◽  
Vol 8 (3) ◽  
pp. e59861 ◽  
Author(s):  
Jared M. Fraser ◽  
Sarah E. Tulk ◽  
Jennifer A. Jeans ◽  
Douglas A. Campbell ◽  
Thomas S. Bibby ◽  
...  
Keyword(s):  
Synechococcus Elongatus ◽  
Iron Starvation ◽  
Synechococcus Elongatus Pcc 7942 ◽  
Pcc 7942 ◽  
Synechocystis Sp ◽  
Pcc 6803

Stability of the Synechococcus elongatus PCC 7942 circadian clock under directed anti-phase expression of the kai genes

Microbiology ◽  
2005 ◽  
Vol 151 (8) ◽  
pp. 2605-2613 ◽  
Author(s):  
Jayna L. Ditty ◽  
Shannon R. Canales ◽  
Breanne E. Anderson ◽  
Stanly B. Williams ◽  
Susan S. Golden
Keyword(s):  
Circadian Clock ◽  
Expression Patterns ◽  
Phase Relationship ◽  
Synechococcus Elongatus ◽  
Cycle Period ◽  
Rhythmic Expression ◽  
Core Components ◽  
Synechococcus Elongatus Pcc 7942 ◽  
Time Required ◽  
Pcc 7942

The kaiA, kaiB and kaiC genes encode the core components of the cyanobacterial circadian clock in Synechococcus elongatus PCC 7942. Rhythmic expression patterns of kaiA and of the kaiBC operon normally peak in synchrony. In some mutants the relative timing of peaks (phase relationship) between these transcription units is altered, but circadian rhythms persist robustly. In this study, the importance of the transcriptional timing of kai genes was examined. Expressing either kaiA or kaiBC from a heterologous promoter whose peak expression occurs 12 h out of phase from the norm, and thus 12 h out of phase from the other kai locus, did not affect the time required for one cycle (period) or phase of the circadian rhythm, as measured by bioluminescence reporters. Furthermore, the data confirm that specific cis elements within the promoters of the kai genes are not necessary to sustain clock function.

scholarly journals Role of KaiC phosphorylation in the circadian clock system of Synechococcus elongatus PCC 7942

2004 ◽  
Vol 101 (38) ◽  
pp. 13927-13932 ◽  
Author(s):  
T. Nishiwaki ◽  
Y. Satomi ◽  
M. Nakajima ◽  
C. Lee ◽  
R. Kiyohara ◽  
...  
Keyword(s):  
Circadian Clock ◽  
Synechococcus Elongatus ◽  
Clock System ◽  
Synechococcus Elongatus Pcc 7942 ◽  
Pcc 7942

scholarly journals CikA, an Input Pathway Component, Senses the Oxidized Quinone Signal to Generate Phase Delays in the Cyanobacterial Circadian Clock

2020 ◽  
Vol 35 (3) ◽  
pp. 227-234 ◽  
Author(s):  
Pyonghwa Kim ◽  
Brianna Porr ◽  
Tetsuya Mori ◽  
Yong-Sung Kim ◽  
Carl H. Johnson ◽  
...  
Keyword(s):  
Circadian Clock ◽  
Time Of Day ◽  
Fine Tuning ◽  
Circadian Oscillation ◽  
Clock Proteins ◽  
Entrainment Process ◽  
Central Oscillator ◽  
External Signals ◽  
Full Synchronization

The circadian clock is a timekeeping system in most organisms that keeps track of the time of day. The rhythm generated by the circadian oscillator must be constantly synchronized with the environmental day/night cycle to make the timekeeping system truly advantageous. In the cyanobacterial circadian clock, quinone is a biological signaling molecule used for entraining and fine-tuning the oscillator, a process in which the external signals are transduced into biological metabolites that adjust the phase of the circadian oscillation. Among the clock proteins, the pseudo-receiver domain of KaiA and CikA can sense external cues by detecting the oxidation state of quinone, a metabolite that reflects the light/dark cycle, although the molecular mechanism is not fully understood. Here, we show the antagonistic phase shifts produced by the quinone sensing of KaiA and CikA. We introduced a new cyanobacterial circadian clock mixture that includes an input component in vitro. KaiA and CikA cause phase advances and delays, respectively, in this circadian clock mixture in response to the quinone signal. In the entrainment process, oxidized quinone modulates the functions of KaiA and CikA, which dominate alternatively at day and night in the cell. This in turn changes the phosphorylation state of KaiC—the central oscillator in cyanobacteria—ensuring full synchronization of the circadian clock. Moreover, we reemphasize the mechanistic input functionality of CikA, contrary to other reports that focus only on its output action.

scholarly journals Real-Time In Vitro Fluorescence Anisotropy of the Cyanobacterial Circadian Clock

2019 ◽  
Vol 2 (2) ◽  
pp. 42 ◽  
Author(s):  
Joel Heisler ◽  
Archana Chavan ◽  
Yong-Gang Chang ◽  
Andy LiWang
Keyword(s):  
Circadian Clock ◽  
Real Time ◽  
Fluorescence Anisotropy ◽  
Polyacrylamide Gel Electrophoresis ◽  
Live Cells ◽  
Ex Post ◽  
Clock Proteins ◽  
Ex Post Facto ◽  
Clock Reaction

Uniquely, the circadian clock of cyanobacteria can be reconstructed outside the complex milieu of live cells, greatly simplifying the investigation of a functioning biological chronometer. The core oscillator component is composed of only three proteins, KaiA, KaiB, and KaiC, and together with ATP they undergo waves of assembly and disassembly that drive phosphorylation rhythms in KaiC. Typically, the time points of these reactions are analyzed ex post facto by denaturing polyacrylamide gel electrophoresis, because this technique resolves the different states of phosphorylation of KaiC. Here, we describe a more sensitive method that allows real-time monitoring of the clock reaction. By labeling one of the clock proteins with a fluorophore, in this case KaiB, the in vitro clock reaction can be monitored by fluorescence anisotropy on the minutes time scale for weeks.

scholarly journals Biological and Structural Basis for Aha1 Regulation of Hsp90 ATPase Activity in Maintaining Proteostasis in the Human Disease Cystic Fibrosis

2010 ◽  
Vol 21 (6) ◽  
pp. 871-884 ◽  
Author(s):  
Atanas V. Koulov ◽  
Paul LaPointe ◽  
Bingwen Lu ◽  
Abbas Razvi ◽  
Judith Coppinger ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Atpase Activity ◽  
Protein Misfolding ◽  
Atp Hydrolysis ◽  
Structural Basis ◽  
Inherited Disease ◽  
Hsp90 Chaperone ◽  
Terminal Domains

The activator of Hsp90 ATPase 1, Aha1, has been shown to participate in the Hsp90 chaperone cycle by stimulating the low intrinsic ATPase activity of Hsp90. To elucidate the structural basis for ATPase stimulation of human Hsp90 by human Aha1, we have developed novel mass spectrometry approaches that demonstrate that the N- and C-terminal domains of Aha1 cooperatively bind across the dimer interface of Hsp90 to modulate the ATP hydrolysis cycle and client activity in vivo. Mutations in both the N- and C-terminal domains of Aha1 impair its ability to bind Hsp90 and stimulate its ATPase activity in vitro and impair in vivo the ability of the Hsp90 system to modulate the folding and trafficking of wild-type and variant (ΔF508) cystic fibrosis transmembrane conductance regulator (CFTR) responsible for the inherited disease cystic fibrosis (CF). We now propose a general model for the role of Aha1 in the Hsp90 ATPase cycle in proteostasis whereby Aha1 regulates the dwell time of Hsp90 with client. We suggest that Aha1 activity integrates chaperone function with client folding energetics by modulating ATPase sensitive N-terminal dimer structural transitions, thereby protecting transient folding intermediates in vivo that could contribute to protein misfolding systems disorders such as CF when destabilized.

scholarly journals Mutations in Novel Lipopolysaccharide Biogenesis Genes Confer Resistance to Amoebal Grazing in Synechococcus elongatus

2016 ◽  
Vol 82 (9) ◽  
pp. 2738-2750 ◽  
Author(s):  
Ryan Simkovsky ◽  
Emily E. Effner ◽  
Maria José Iglesias-Sánchez ◽  
Susan S. Golden
Keyword(s):  
Biomass Production ◽  
Lipid A ◽  
Synechococcus Elongatus ◽  
Cyanobacterial Blooms ◽  
Core Oligosaccharide ◽  
Diverse Range ◽  
Content Type ◽  
O Antigen ◽  
Population Structures ◽  
Pcc 7942

ABSTRACTIn natural and artificial aquatic environments, population structures and dynamics of photosynthetic microbes are heavily influenced by the grazing activity of protistan predators. Understanding the molecular factors that affect predation is critical for controlling toxic cyanobacterial blooms and maintaining cyanobacterial biomass production ponds for generating biofuels and other bioproducts. We previously demonstrated that impairment of the synthesis or transport of the O-antigen component of lipopolysaccharide (LPS) enables resistance to amoebal grazing in the model predator-prey system consisting of the heterolobosean amoeba HGG1 and the cyanobacteriumSynechococcus elongatusPCC 7942 (R. S. Simkovsky et al., Proc Natl Acad Sci U S A 109:16678–16683, 2012,http://dx.doi.org/10.1073/pnas.1214904109). In this study, we used this model system to identify additional gene products involved in the synthesis of O antigen, the ligation of O antigen to the lipid A-core conjugated molecule (including a novel ligase gene), the generation of GDP-fucose, and the incorporation of sugars into the lipid A core oligosaccharide ofS. elongatus. Knockout of any of these genes enables resistance to HGG1, and of these, only disruption of the genes involved in synthesis or incorporation of GDP-fucose into the lipid A-core molecule impairs growth. Because these LPS synthesis genes are well conserved across the diverse range of cyanobacteria, they enable a broader understanding of the structure and synthesis of cyanobacterial LPS and represent mutational targets for generating resistance to amoebal grazers in novel biomass production strains.

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