Evolutionary Innovation by Polyploidy

The preferred conditions for evolutionary innovation represent a fundamental question, but little is known experimentally or theoretically. In this study, we focused on the potential role of polyploidy in the evolution of novel traits. We proposed a simple model and demonstrated that the evolutionary rate of polyploids is similar to more much slower than that of haploids under neutral selection or during gradual evolution. However, experiments using polyploid cyanobacteria demonstrated that the probability of achieving antibiotic resistance increased with the number of chromosomes and implied an optimal number of chromosomes. Then, we investigated the dynamics of the same model on a fitness landscape in which cells should jump over a lethal valley to increase their fitness. The evolutionary rate could be increased in polyploidy, and the optimal number of chromosomes was identified. Further, we proposed that the optimization for evolutionary innovation might determine the number of chromosomes in polyploid bacteria.Comment: 35 pages, 8 figures, 4 table

Discovery of novel replication proteins for large plasmids in cyanobacteria and their potential applications in genetic engineering

Numerous cyanobacteria capable of oxygenic photosynthesis possess multiple large plasmids exceeding 100 kbp in size. These plasmids are believed to have distinct replication and distribution mechanisms, as they coexist within cells without causing incompatibilities between plasmids. However, information on plasmid replication proteins (Rep) in cyanobacteria is limited. Synechocystis sp. PCC 6803 hosts four large plasmids, pSYSM, pSYSX, pSYSA, and pSYSG, but Rep proteins for these plasmids, except for CyRepA1 on pSYSA, are unknown. Using Autonomous Replication sequencing (AR-seq), we identified two potential Rep genes in Synechocystis 6803, slr6031 and slr6090, both located on pSYSX. The corresponding Rep candidates, Slr6031 and Slr6090, share structural similarities with Rep-associated proteins of other bacteria and homologs were also identified in various cyanobacteria. We observed autonomous replication activity for Slr6031 and Slr6090 in Synechococcus elongatus PCC 7942 by fusing their genes with a construct expressing GFP and introducing them via transformation. The slr6031/slr6090-containing plasmids exhibited lower copy numbers and instability in Synechococcus 7942 cells compared to the expression vector pYS. While recombination occurred in the case of slr6090, the engineered plasmid with slr6031 coexisted with plasmids encoding CyRepA1 or Slr6090 in Synechococcus 7942 cells, indicating the compatibility of Slr6031 and Slr6090 with CyRepA1. Based on these results, we designated Slr6031 and Slr6090 as CyRepX1 (Cyanobacterial Rep-related protein encoded on pSYSX) and CyRepX2, respectively, demonstrating that pSYSX is a plasmid with “two Reps in one plasmid.” Furthermore, we determined the copy number and stability of plasmids with cyanobacterial Reps in Synechococcus 7942 and Synechocystis 6803 to elucidate their potential applications. The novel properties of CyRepX1 and 2, as revealed by this study, hold promise for the development of innovative genetic engineering tools in cyanobacteria

Glycogen deficiency enhances carbon partitioning into glutamate for an alternative extracellular metabolic sink in cyanobacteria

Abstract Glycogen serves as a metabolic sink in cyanobacteria. Glycogen deficiency causes the extracellular release of distinctive metabolites such as pyruvate and 2-oxoglutarate upon nitrogen depletion; however, the mechanism has not been fully elucidated. This study aimed to elucidate the mechanism of carbon partitioning in glycogen-deficient cyanobacteria. Extracellular and intracellular metabolites in a glycogen-deficient ΔglgC mutant of Synechococcus elongatus PCC 7942 were comprehensively analyzed. In the presence of a nitrogen source, the ΔglgC mutant released extracellular glutamate rather than pyruvate and 2-oxoglutarate, whereas its intracellular glutamate level was lower than that in the wild-type strain. The de novo synthesis of glutamate increased in the ΔglgC mutant, suggesting that glycogen deficiency enhanced carbon partitioning into glutamate and extracellular excretion through an unidentified transport system. This study proposes a model in which glutamate serves as the prime extracellular metabolic sink alternative to glycogen when nitrogen is available

Coordination of Polyploid Chromosome Replication with Cell Size and Growth in a Cyanobacterium

Polyploidy has evolved many times across the kingdom of life. The relationship between cell growth and chromosome replication in bacteria has been studied extensively in monoploid model organisms such as Escherichia coli but not in polyploid organisms. Our study of the polyploid cyanobacterium Synechococcus elongatus demonstrates that replicating chromosome number is restricted and regulated by DnaA to maintain a relatively stable gene copy number/cell volume ratio during cell growth. In addition, our results suggest that polyploidy confers resistance to UV, which damages DNA. This compensatory polyploidy is likely necessitated by photosynthesis, which requires sunlight and generates damaging reactive oxygen species, and may also explain how polyploid bacteria can adapt to extreme environments with high risk of DNA damage.Homologous chromosome number (ploidy) has diversified among bacteria, archaea, and eukaryotes over evolution. In bacteria, model organisms such as Escherichia coli possess a single chromosome encoding the entire genome during slow growth. In contrast, other bacteria, including cyanobacteria, maintain multiple copies of individual chromosomes (polyploid). Although a correlation between ploidy level and cell size has been observed in bacteria and eukaryotes, it is poorly understood how replication of multicopy chromosomes is regulated and how ploidy level is adjusted to cell size. In addition, the advantages conferred by polyploidy are largely unknown. Here we show that only one or a few multicopy chromosomes are replicated at once in the cyanobacterium Synechococcus elongatus and that this restriction depends on regulation of DnaA activity. Inhibiting the DnaA intrinsic ATPase activity in S. elongatus increased the number of replicating chromosomes and chromosome number per cell but did not affect cell growth. In contrast, when cell growth rate was increased or decreased, DnaA level, DnaA activity, and the number of replicating chromosomes also increased or decreased in parallel, resulting in nearly constant chromosome copy number per unit of cell volume at constant temperature. When chromosome copy number was increased by inhibition of DnaA ATPase activity or reduced culture temperature, cells exhibited greater resistance to UV light. Thus, it is suggested that the stepwise replication of the genome enables cyanobacteria to maintain nearly constant gene copy number per unit of cell volume and that multicopy chromosomes function as backup genetic information to compensate for genomic damage

ppGpp accumulation reduces the expression of the global nitrogen homeostasis-modulating NtcA regulon by affecting 2-oxoglutarate levels

Abstract The cyanobacterium Synechococcus elongatus PCC 7942 accumulates alarmone guanosine tetraphosphate (ppGpp) under stress conditions, such as darkness. A previous study observed that artificial ppGpp accumulation under photosynthetic conditions led to the downregulation of genes involved in the nitrogen assimilation system, which is activated by the global nitrogen regulator NtcA, suggesting that ppGpp regulates NtcA activity. However, the details of this mechanism have not been elucidated. Here, we investigate the metabolic responses associated with ppGpp accumulation by heterologous expression of the ppGpp synthetase RelQ. The pool size of 2-oxoglutarate (2-OG), which activates NtcA, is significantly decreased upon ppGpp accumulation. De novo 13C-labeled CO2 assimilation into the Calvin-Benson-Bassham cycle and glycolytic intermediates continues irrespective of ppGpp accumulation, whereas the labeling of 2-OG is significantly decreased under ppGpp accumulation. The low 2-OG levels in the RelQ overexpression cells could be because of the inhibition of metabolic enzymes, including aconitase, which are responsible for 2-OG biosynthesis. We propose a metabolic rearrangement by ppGpp accumulation, which negatively regulates 2-OG levels to maintain carbon and nitrogen balance

Intensive DNA Replication and Metabolism during the Lag Phase in Cyanobacteria

<div><p>Unlike bacteria such as <i>Escherichia coli</i> and <i>Bacillus subtilis</i>, 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 <i>Synechococcus elongatus</i> PCC 7942 (hereafter <i>Synechococcus</i> 7942). In <i>Synechococcus</i> 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 <i>Synechococcus</i> 7942. To address this hypothesis, we analysed the relationship between DNA replication and cell doubling at various growth phases of <i>Synechococcus</i> 7942 cell culture. Three distinct growth phases were characterised in <i>Synechococcus</i> 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 <i>Synechococcus</i> 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 <i>Synechococcus</i> 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.</p></div

Metabolite profiles of <i>Synechococcus</i> 7942 during batch culture.

<p>The stationary culture of <i>Synechococcus</i> 7942 wild-type was diluted to OD₇₅₀ = 0.2 in fresh BG11 medium. After 18 hr in the dark, cultures were transferred to illuminated conditions (time 0). Samples were harvested at various times points post-transfer. The absolute amounts (pmol per 10⁷ cells) of metabolites were determined at each time point with CE-TOFMS. The data show remarkable changes at each phase in central carbon metabolites, TCA cycle metabolites, amino acids, purines, and pyrimidines. Raw data and additional figures are included in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136800#pone.0136800.s007" target="_blank">S2 Table</a>, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136800#pone.0136800.s002" target="_blank">S2</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136800#pone.0136800.s005" target="_blank">S5</a> Figs.</p

Growth and chromosome copy number during long-term culture.

<p>(A) Growth curve of <i>Synechococcus</i> 7942^TK after transfer to illuminated conditions, with a diagram showing the culture strategy. The stationary cultures were diluted to OD₇₅₀ = 0.2 in fresh BG11 liquid medium. After 18 hr in the dark, cultures were transferred to light conditions (time 0). (B) DNA content profiles of <i>Synechococcus</i> 7942^TK cells. Cells were harvested at each time point and analysed with flow cytometry. (C) Expression of RpoD1, DnaK2, and HA-tagged thymidine kinase (HA-TK). <i>Synechococcus</i> 7942^TK cultures were harvested at the indicated times after transfer to liquid medium, and crude extracts were prepared. Samples containing 5 μg (RpoD1 and DnaK2) or 20 μg (HA-TK) of total protein were analysed with western blot using the appropriate antibodies.</p