Observation of Photobehavior in <em>Chlamydomonas reinhardtii</em>

For the survival of the motile phototrophic microorganisms, being under proper light conditions is crucial. Consequently, they show photo-induced ...

Reactivation of Demembranated Cell Models in <em>Chlamydomonas reinhardtii</em>

Since the historical experiment on the contraction of glycerinated muscle by adding ATP, which Szent-Gy&#246;rgyi demonstrated in the mid-20th century, in ...

Tokyo Institute of Technology

The ATPase activity of chloroplast and bacterial F(1)-ATPase is strongly inhibited by both the endogenous inhibitor ε and tightly bound ADP. Although the physiological significance of these inhibitory mechanisms is not very well known for the membrane-bound F(0)F(1), these are very likely to be important in avoiding the futile ATP hydrolysis reaction and ensuring efficient ATP synthesis in vivo. In a previous study using the α(3)β(3)γ complex of F(1) obtained from the thermophilic cyanobacteria, Thermosynechococcus elongatus BP-1, we succeeded in determining the discrete stop position, ∼80° forward from the pause position for ATP binding, caused by ε-induced inhibition (ε-inhibition) during γ rotation (Konno, H., Murakami-Fuse, T., Fujii, F., Koyama, F., Ueoka-Nakanishi, H., Pack, C. G., Kinjo, M., and Hisabori, T. (2006) EMBO J. 25, 4596-4604). Because γ in ADP-inhibited F(1) also pauses at the same position, ADP-induced inhibition (ADP-inhibition) was assumed to be linked to ε-inhibition. However, ADP-inhibition and ε-inhibition should be independent phenomena from each other because the ATPase core complex, α(3)β(3)γ, also lapses into the ADP-inhibition state. By way of thorough biophysical and biochemical analyses, we determined that the ε subunit inhibition mechanism does not directly correlate with ADP-inhibition. We suggest here that the cyanobacterial ATP synthase ε subunit carries out an important regulatory role in acting as an independent "braking system" for the physiologically unfavorable ATP hydrolysis reaction.

Characterization of the relationship between ADP- and epsilon-induced inhibition in cyanobacterial F1-ATPase.

The diazotrophic cyanobacterium Anabaena sp. strain PCC 7120 (A.7120) differentiates into specialized heterocyst cells that fix nitrogen under nitrogen starvation conditions. Although reducing equivalents are essential for nitrogen fixation, little is known about redox systems in heterocyst cells. In this study, we investigated thioredoxin (Trx) networks in Anabaena using TrxM, and identified 16 and 38 candidate target proteins in heterocysts and vegetative cells, respectively, by Trx affinity chromatography (Motohashi et al. (Comprehensive survey of proteins targeted by chloroplast thioredoxin. Proc Natl Acad Sci USA, 2001; 98: , 11224-11229)). Among these, the Fe-S cluster scaffold protein NifU that facilitates functional expression of nitrogenase in heterocysts was found to be a potential TrxM target. Subsequently, we observed that the scaffold activity of N-terminal catalytic domain of NifU is enhanced in the presence of Trx-system, suggesting that TrxM is involved in the Fe-S cluster biogenesis. 

Involvement of thioredoxin on the scaffold activity of NifU in heterocyst cells of the diazotrophic cyanobacterium Anabaena sp. strain PCC 7120.

In the last decade, many studies have been conducted to employ genetically engineered cyanobacteria in the production of various metabolites. However, the lack of a strict gene regulation system in cyanobacteria has hampered these attempts. The filamentous cyanobacterium Anabaena sp. PCC 7120 performs both nitrogen and carbon fixation and is, therefore, a good candidate organism for such production. To employ Anabaena cells for this purpose, we intended to develop artificial gene regulation systems to alter the cell metabolic pathways efficiently. We introduced into Anabaena a transcriptional repressor TetR, widely used in diverse organisms, and green fluorescent protein (GFP) as a reporter. We found that anhydrotetracycline (aTc) substantially induced GFP fluorescence in a concentration-dependent manner. By expressing tetR under the nitrate-specific promoter nirA, we successfully reduced the concentration of aTc required for the induction of gfp under nitrogen fixation conditions (to 10% of the concentration needed under nitrate-replete conditions). Further, we succeeded in the overexpression of GFP by depletion of nitrate without the inducer by means of promoter engineering of the nirA promoter. Moreover, we applied these gene regulation systems to a metabolic enzyme in Anabaena and successfully repressed glnA, the gene encoding glutamine synthetase that is essential for nitrogen assimilation in cyanobacteria, by expressing the small antisense RNA for glnA. Consequently, the ammonium production of an ammonium-excreting Anabaena mutant was significantly enhanced. We therefore conclude that the gene regulation systems developed in this study are useful tools for the regulation of metabolic enzymes and will help to increase the production of desired substances in Anabaena. 

Efficient Gene Induction and Endogenous Gene Repression Systems for the Filamentous Cyanobacterium Anabaena sp. PCC 7120.

In recent years, studies on the development of gene regulation tools in cyanobacteria have been extensively conducted toward efficient production of valuable chemicals. However, there is considerable scope for improving the economic feasibility of production. To improve a recently reported gene induction system using anhydrotetracycline (aTc)-TetR and an endogenous gene repression system using small antisense RNA in the filamentous nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120 (Anabaena), we constructed a positive feedback loop, in which gfp and a small antisense RNA for tetR are controlled by an aTc-inducible promoter. GFP expression in this improved system was higher and longer than the system lacking tetR repression. In addition, by using TetR aptamer and a riboswitch, we succeeded in achieving a superior and longer induction of GFP expression even under high-light conditions. Hence, efficient gene induction systems were established in Anabaena by designing a gene regulation network using RNA-based tools.

Designing Synthetic Flexible Gene Regulation Networks Using RNA Devices in Cyanobacteria.

In the last decade, much progress has been made in the photosynthetic production of valuable products using unicellular cyanobacteria. However, production of some products requires dark, anaerobic incubation, which prevents practical applications using these organisms. Anabaena sp. PCC 7120 (A. 7120) is a heterocyst-forming multicellular cyanobacterium that is easy to manipulate genetically. Upon nitrogen step-down, this strain differentiates heterocysts that retain micro-oxic conditions for nitrogen fixation. We have developed gene regulation tools in this cyanobacterium. However, lack of a cell type-specific gene induction system has prevented A. 7120 from becoming a bona fide attractive host for photosynthetic production. We validated the usability of two transcriptional ON riboswitches that respond to theophylline or adenine. We then created a cell type-specific gene induction system by combining the riboswitches and promoters specific to either heterocysts or vegetative cells. We also created another cell type-specific gene induction system using small RNA that activates translation. Consequently, our study has expanded the toolbox for gene regulation in cyanobacteria and has enabled spatio-temporal gene induction in multicellular cyanobacteria.

Spatio-Temporal Gene Induction Systems in the Heterocyst-Forming Multicellular Cyanobacterium Anabaena sp. PCC 7120.

Anabaena sp. PCC 7120 (A. 7120) is a heterocyst-forming multicellular cyanobacterium that performs nitrogen fixation. This cyanobacterium has been extensively studied as a model for multicellularity in prokaryotic cells. We have been interested in photosynthetic production of nitrogenous compounds using A. 7120. However, the lack of efficient gene repression tools has limited its usefulness. We originally developed an artificial endogenous gene repression method in this cyanobacterium using small antisense RNA. However, the narrow dynamic range of repression of this method needs to be improved. Recently, clustered regularly interspaced short palindromic repeat (CRISPR) interference (CRISPRi) technology was developed and was successfully applied in some unicellular cyanobacteria. The technology requires expression of nuclease-deficient CRISPR-associated protein 9 (dCas9) and a single guide RNA (sgRNA) that is complementary to a target sequence, to repress expression of the target gene. In this study, we employed CRISPRi technology for photosynthetic production of ammonium through repression of glnA, the only gene encoding glutamine synthetase that is essential for nitrogen assimilation in A. 7120. By strictly regulating dCas9 expression using the TetR gene induction system, we succeeded in fine-tuning the GlnA protein in addition to the level of glnA transcripts. Expression of sgRNA by the heterocyst-specific nifB promoter led to efficient repression of GlnA in heterocysts, as well as in vegetative cells. Finally, we showed that ammonium is excreted into the medium only when inducers of expression of dCas9 were added. In conclusion, CRISPRi enables temporal control of desired products and will be a useful tool for basic science.

Application of CRISPR Interference for Metabolic Engineering of the Heterocyst-Forming Multicellular Cyanobacterium Anabaena sp. PCC 7120.

F is a soluble part of FF-ATP synthase and performs a catalytic process of ATP hydrolysis and synthesis. The γ subunit, which is the rotary shaft of F motor, is composed of N-terminal and C-terminal helices domains, and a protruding Rossman-fold domain located between the two major helices parts. The N-terminal and C-terminal helices domains of γ assemble into an antiparallel coiled-coil structure, and are almost embedded into the stator ring composed of αβ hexamer of the F molecule. Cyanobacterial and chloroplast γ subunits harbor an inserted sequence of 30 or 39 amino acids length within the Rossman-fold domain in comparison with bacterial or mitochondrial γ. To understand the structure-function relationship of the γ subunit, we prepared a mutant F-ATP synthase of a thermophilic cyanobacterium, Thermosynechococcus elongatus BP-1, in which the γ subunit is split into N-terminal α-helix along with the inserted sequence and the remaining C-terminal part. The obtained mutant showed higher ATP-hydrolysis activities than those containing the wild-type γ. Contrary to our expectation, the complexes containing the split γ subunits were mostly devoid of the C-terminal helix. We further investigated the effect of post-assembly cleavage of the γ subunit. We demonstrate that insertion of the nick between two helices of the γ subunit imparts resistance to ADP inhibition, and the C-terminal α-helix is dispensable for ATP-hydrolysis activity and plays a crucial role in the assembly of F-ATP synthase.

Amputation of a C-terminal helix of the γ subunit increases ATP-hydrolysis activity of cyanobacterial F ATP synthase.

Channelrhodopsins (ChRs) are the light-gated ion channels that have opened the research field of optogenetics. They were originally identified in the green alga Chlamydomonas reinhardtii, a biciliated unicellular alga that inhabits in freshwater, swims with the cilia, and undergoes photosynthesis. It has various advantages as an experimental organism and is used in a wide range of research fields including photosynthesis, cilia, and sexual reproduction. ChRs function as the primary photoreceptor for the cell's photo-behavioral responses, seen as changes in the manner of swimming after photoreception. In this chapter, we will introduce C. reinhardtii as an experimental organism and explain our current understanding of how the cell senses light and shows photo-behavioral responses.

Atsuko Isu

Laboratory for Chemistry and Life Science,
Institute of Innovative Research,
Laboratory for Chemistry and Life Science, Institute of Innovative Research

Tokyo Institute of Technology

Observation of Photobehavior in Chlamydomonas reinhardtii

Reactivation of Demembranated Cell Models in Chlamydomonas reinhardtii

Atsuko Isu

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Laboratory for Chemistry and Life Science,Institute of Innovative Research,Laboratory for Chemistry and Life Science, Institute of Innovative Research

Tokyo Institute of Technology

Observation of Photobehavior in Chlamydomonas reinhardtii

Reactivation of Demembranated Cell Models in Chlamydomonas reinhardtii

Laboratory for Chemistry and Life Science,
Institute of Innovative Research,
Laboratory for Chemistry and Life Science, Institute of Innovative Research