Ryudo Ohbayashi

Intensive DNA Replication and Metabolism during the Lag Phase in Cyanobacteria

Unlike bacteria such as Escherichia coli and Bacillus subtilis, several species of freshwater cyanobacteria are known to contain multiple chromosomal copies per cell, at all stages of their cell cycle. We have characterized the replication of multi-copy chromosomes in the cyanobacterium Synechococcus elongatus PCC 7942 (hereafter Synechococcus 7942). In Synechococcus 7942, the replication of multi-copy chromosome is asynchronous, not only among cells but also among multi-copy chromosomes. This suggests that DNA replication is not tightly coupled to cell division in Synechococcus 7942. To address this hypothesis, we analysed the relationship between DNA replication and cell doubling at various growth phases of Synechococcus 7942 cell culture. Three distinct growth phases were characterised in Synechococcus 7942 batch culture: lag phase, exponential phase, and arithmetic (linear) phase. The chromosomal copy number was significantly higher during the lag phase than during the exponential and linear phases. Likewise, DNA replication activity was higher in the lag phase cells than in the exponential and linear phase cells, and the lag phase cells were more sensitive to nalidixic acid, a DNA gyrase inhibitor, than cells in other growth phases. To elucidate physiological differences in Synechococcus 7942 during the lag phase, we analysed the metabolome at each growth phase. In addition, we assessed the accumulation of central carbon metabolites, amino acids, and DNA precursors at each phase. The results of these analyses suggest that Synechococcus 7942 cells prepare for cell division during the lag phase by initiating intensive chromosomal DNA replication and accumulating metabolites necessary for the subsequent cell division and elongation steps that occur during the exponential growth and linear phases.

A tightly inducible riboswitch system in Synechocystis sp. PCC 6803

Cyanobacteria are photosynthetic microorganisms that serve as experimental model organisms for the study of photosynthesis, environmental stress responses, and the production of biofuels. Genetic tools for bioengineering have been developed as a result of such studies. However, there is still room for improvement for the tight control of experimental protein expression in these microorganisms. Here, we describe an expression system controlled by a theophylline-responsive riboswitch that we have constructed in the cyanobacterium Synechocystis sp. PCC 6803. We demonstrate that, in response to different theophylline concentrations, this riboswitch can tightly control green fluorescence protein expression in Synechocystis. Thus, this system is useful as a tool for genetic engineering and the synthetic biology of cyanobacteria.

Acidophilic green algal genome provides insights into adaptation to an acidic environment

Significance Extremely acidic environments are scattered worldwide, and their ecosystems are supported by acidophilic microalgae as primary producers. To understand how acidophilic algae evolved from their respective neutrophilic ancestors, we determined the draft genome sequence of the acidophilic green alga Chlamydomonas eustigma and performed comparative genome analyses between C. eustigma and its neutrophilic relative Chlamydomonas reinhardtii. The results suggest that higher expression of heat-shock proteins and H+-ATPase, loss of some metabolic pathways that acidify cytosol, and acquisition of metal-detoxifying genes by horizontal gene transfer have played important roles in the adaptation to acidic environments. These features are also found in other acidophilic green and red algae, suggesting the existence of common mechanisms in the adaptation to acidic environments. Some microalgae are adapted to extremely acidic environments in which toxic metals are present at high levels. However, little is known about how acidophilic algae evolved from their respective neutrophilic ancestors by adapting to particular acidic environments. To gain insights into this issue, we determined the draft genome sequence of the acidophilic green alga Chlamydomonas eustigma and performed comparative genome and transcriptome analyses between C. eustigma and its neutrophilic relative Chlamydomonas reinhardtii. The results revealed the following features in C. eustigma that probably contributed to the adaptation to an acidic environment. Genes encoding heat-shock proteins and plasma membrane H+-ATPase are highly expressed in C. eustigma. This species has also lost fermentation pathways that acidify the cytosol and has acquired an energy shuttle and buffering system and arsenic detoxification genes through horizontal gene transfer. Moreover, the arsenic detoxification genes have been multiplied in the genome. These features have also been found in other acidophilic green and red algae, suggesting the existence of common mechanisms in the adaptation to acidic environments.

Diversification of DnaA dependency for DNA replication in cyanobacterial evolution.

Regulating DNA replication is essential for all living cells. The DNA replication initiation factor DnaA is highly conserved in prokaryotes and is required for accurate initiation of chromosomal replication at oriC. DnaA-independent free-living bacteria have not been identified. The dnaA gene is absent in plastids and some symbiotic bacteria, although it is not known when or how DnaA-independent mechanisms were acquired. Here, we show that the degree of dependency of DNA replication on DnaA varies among cyanobacterial species. Deletion of the dnaA gene in Synechococcus elongatus PCC 7942 shifted DNA replication from oriC to a different site as a result of the integration of an episomal plasmid. Moreover, viability during the stationary phase was higher in dnaA disruptants than in wild-type cells. Deletion of dnaA did not affect DNA replication or cell growth in Synechocystis sp. PCC 6803 or Anabaena sp. PCC 7120, indicating that functional dependency on DnaA was already lost in some nonsymbiotic cyanobacterial lineages during diversification. Therefore, we proposed that cyanobacteria acquired DnaA-independent replication mechanisms before symbiosis and such an ancestral cyanobacterium was the sole primary endosymbiont to form a plastid precursor.

Development of a Double Nuclear Gene-Targeting Method by Two-Step Transformation Based on a Newly Established Chloramphenicol-Selection System in the Red Alga Cyanidioschyzon merolae

The unicellular red alga Cyanidioschyzon merolae possesses a simple cellular architecture that consists of one mitochondrion, one chloroplast, one peroxisome, one Golgi apparatus, and several lysosomes. The nuclear genome content is also simple, with very little genetic redundancy (16.5 Mbp, 4,775 genes). In addition, molecular genetic tools such as gene targeting and inducible gene expression systems have been recently developed. These cytological features and genetic tractability have facilitated various omics analyses. However, only a single transformation selection marker URA has been made available and thus the application of genetic modification has been limited. Here, we report the development of a nuclear targeting method by using chloramphenicol and the chloramphenicol acetyltransferase (CAT) gene. In addition, we found that at least 200-bp homologous arms are required and 500-bp arms are sufficient for a targeted single-copy insertion of the CAT selection marker into the nuclear genome. By means of a combination of the URA and CAT transformation systems, we succeeded in producing a C. merolae strain that expresses HA-cyclin 1 and FLAG-CDKA from the chromosomal CYC1 and CDKA loci, respectively. These methods of multiple nuclear targeting will facilitate genetic manipulation of C. merolae.

Variety of DNA Replication Activity Among Cyanobacteria Correlates with Distinct Respiration Activity in the Dark

Cyanobacteria exhibit light-dependent cell growth since most of their cellular energy is obtained by photosynthesis. In Synechococcus elongatus PCC 7942, one of the model cyanobacteria, DNA replication depends on photosynthetic electron transport. However, the critical signal for the regulatory mechanism of DNA replication has not been identified. In addition, conservation of this regulatory mechanism has not been investigated among cyanobacteria. To understand this regulatory signal and its dependence on light, we examined the regulation of DNA replication under both light and dark conditions among three model cyanobacteria, S. elongatus PCC 7942, Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120. Interestingly, DNA replication activity in Synechocystis and Anabaena was retained when cells were transferred to the dark, although it was drastically decreased in S. elongatus. Glycogen metabolism and respiration were higher in Synechocystis and Anabaena than in S. elongatus in the dark. Moreover, DNA replication activity in Synechocystis and Anabaena was reduced to the same level as that in S. elongatus by inhibition of respiratory electron transport after transfer to the dark. These results demonstrate that there is disparity in DNA replication occurring in the dark among cyanobacteria, which is caused by the difference in activity of respiratory electron transport.

ParA-like protein influences the distribution of multi-copy chromosomes in cyanobacterium Synechococcus elongatus PCC 7942

While many bacteria, such as Escherichia coli and Bacillus subtilis, harbour a single-copy chromosome, freshwater cyanobacteria have multiple copies of each chromosome per cell. Although it has been reported that multi-copy chromosomes are evenly distributed along the major axis of the cell in cyanobacterium Synechococcus elongatus PCC 7942, the distribution mechanism of these chromosomes remains unclear. In S. elongatus, the carboxysome, a metabolic microcompartment for carbon fixation that is distributed in a similar manner to the multi-copy chromosomes, is regulated by ParA-like protein (hereafter ParA). To elucidate the role of ParA in the distribution of multi-copy chromosomes, we constructed and analysed ParA disruptant and overexpressing strains of S. elongatus. Our fluorescence in situ hybridization assay revealed that the parA disruptants displayed an aberrant distribution of their multi-copy chromosomes. In the parA disruptant the multiple origin and terminus foci, corresponding to the intracellular position of each chromosomal region, were aggregated, which was compensated by the expression of exogenous ParA from other genomic loci. The parA disruptant is sensitive to UV-C compared to the WT strain. Additionally, giant cells appeared under ParA overexpression at the late stage of growth indicating that excess ParA indirectly inhibits cell division. Screening of the ParA-interacting proteins by yeast two-hybrid analysis revealed four candidates that are involved in DNA repair and cell membrane biogenesis. These results suggest that ParA is involved in the pleiotropic cellular functions with these proteins, while parA is dispensable for cell viability in S. elongatus.