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Journal ArticleDOI

Ploidy in cyanobacteria

01 Oct 2011-Fems Microbiology Letters (FEMS Microbiol Lett)-Vol. 323, Iss: 2, pp 124-131
TL;DR: A recently developed real-time PCR method for the determination of genome copy numbers was optimized for the application to cyanobacteria and revealed that for Synechocystis PCC 6803 strain differences exist and that the ploidy level is highly growth phase-regulated.
Abstract: A recently developed real-time PCR method for the determination of genome copy numbers was optimized for the application to cyanobacteria. Three species were chosen to represent a fresh water species, a salt water species, and two strains of a widely used laboratory species. Synechococcus PCC 7942 and Synechococcus WH7803 were found to contain 3-4 genome copies per cell and are thus oligoploid, confirming earlier publications. In contrast, Synechocystis PCC 6803 is highly polyploid. The motile wild-type strain contains 218 genome copies in exponential phase and 58 genome copies in linear and in stationary growth phase. The GT wild-type strain contains 142 genome copies in exponential phase and 42 genome copies in linear and stationary growth phase. These are the highest numbers found for any cyanobacterial species. Notably these values are much higher than the value of 12 genome copies published for the 'Kazusa' strain more than 20 years ago. The results reveal that for Synechocystis PCC 6803 strain differences exist and that the ploidy level is highly growth phase-regulated. A compilation of the ploidy levels of all investigated cyanobacterial species gives an overview of the genome copy number distribution and shows that monoploid, oligoploid, and polyploid cyanobacteria exist.
Citations
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Journal ArticleDOI
TL;DR: The markerless nature of cpf1 genome editing will allow for complex genome modification that was not possible with previously existing technology while facilitating the development of cyanobacteria as highly modified biofactories.
Abstract: Cyanobacteria are the ideal organisms for the production of a wide range of bioproducts as they can convert CO2 directly into the desired end product using solar energy. Unfortunately, the engineering of cyanobacteria to create efficient cell factories has been impaired by the cumbersome genetic tools that are currently available for these organisms; especially when trying to accumulate multiple modifications. We sought to construct an efficient and precise tool for generating numerous markerless modifications in cyanobacteria using CRISPR technology and the alternative nuclease, Cpf1. In this study we demonstrate rapid engineering of markerless knock-ins, knock-outs and point mutations in each of three model cyanobacteria; Synechococcus, Synechocystis and Anabaena. The markerless nature of cpf1 genome editing will allow for complex genome modification that was not possible with previously existing technology while facilitating the development of cyanobacteria as highly modified biofactories.

229 citations

Journal ArticleDOI
TL;DR: The new biosynthesis routes 'tap' the metabolism ever more efficiently, particularly through the engineering of driving forces and utilization of cofactors generated during the light reactions of photosynthesis, resulting in higher product titers.

221 citations

Journal ArticleDOI
TL;DR: The nuclease-deficient Cas9 from the type-II CRISPR/Cas of Streptrococcus pyogenes was used to repress green fluorescent protein (GFP) to negligible levels and it is demonstrated that tightly repressed promoters allow for inducible and reversible CRISpri in cyanobacteria.
Abstract: We describe the application of clustered regularly interspaced short palindromic repeats interference (CRISPRi) for gene repression in the model cyanobacterium Synechcocystis sp. PCC 6803. The nuclease-deficient Cas9 from the type-II CRISPR/Cas of Streptrococcus pyogenes was used to repress green fluorescent protein (GFP) to negligible levels. CRISPRi was also used to repress formation of carbon storage compounds polyhydroxybutryate (PHB) and glycogen during nitrogen starvation. As an example of the potential of CRISPRi for basic and applied cyanobacteria research, we simultaneously knocked down 4 putative aldehyde reductases and dehydrogenases at 50–95% repression. This work also demonstrates that tightly repressed promoters allow for inducible and reversible CRISPRi in cyanobacteria.

204 citations

Journal ArticleDOI
TL;DR: Results indicate that the introduction of a lactate dehydrogenase rescues this phenotype by preventing the reversion of the ldh mutant, a phenotype which is shown to be stable for prolonged batch culturing.
Abstract: Metabolic engineering of microorganisms has become a versatile tool to facilitate production of bulk chemicals, fuels, etc. Accordingly, CO(2) has been exploited via cyanobacterial metabolism as a sustainable carbon source of biofuel and bioplastic precursors. Here we extended these observations by showing that integration of an ldh gene from Bacillus subtilis (encoding an l-lactate dehydrogenase) into the genome of Synechocystis sp. strain PCC6803 leads to l-lactic acid production, a phenotype which is shown to be stable for prolonged batch culturing. Coexpression of a heterologous soluble transhydrogenase leads to an even higher lactate production rate and yield (lactic acid accumulating up to a several-millimolar concentration in the extracellular medium) than those for the single ldh mutant. The expression of a transhydrogenase alone, however, appears to be harmful to the cells, and a mutant carrying such a gene is rapidly outcompeted by a revertant(s) with a wild-type growth phenotype. Furthermore, our results indicate that the introduction of a lactate dehydrogenase rescues this phenotype by preventing the reversion.

187 citations

Journal ArticleDOI
TL;DR: High expression levels of the Cas9 protein in Synechococcus 2973 appear to be toxic and result in cell death, but introduction of a CRISPR/Cas9 genome editing system on a plasmid backbone that leads to transient cas9 expression allowed for efficient markerless genome editing in a wild type genetic background.
Abstract: As autotrophic prokaryotes, cyanobacteria are ideal chassis organisms for sustainable production of various useful compounds. The newly characterized cyanobacterium Synechococcus elongatus UTEX 2973 is a promising candidate for serving as a microbial cell factory because of its unusually rapid growth rate. Here, we seek to develop a genetic toolkit that enables extensive genomic engineering of Synechococcus 2973 by implementing a CRISPR/Cas9 editing system. We targeted the nblA gene because of its important role in biological response to nitrogen deprivation conditions. First, we determined that the Streptococcus pyogenes Cas9 enzyme is toxic in cyanobacteria, and conjugational transfer of stable, replicating constructs containing the cas9 gene resulted in lethality. However, after switching to a vector that permitted transient expression of the cas9 gene, we achieved markerless editing in 100 % of cyanobacterial exconjugants after the first patch. Moreover, we could readily cure the organisms of antibiotic resistance, resulting in a markerless deletion strain. High expression levels of the Cas9 protein in Synechococcus 2973 appear to be toxic and result in cell death. However, introduction of a CRISPR/Cas9 genome editing system on a plasmid backbone that leads to transient cas9 expression allowed for efficient markerless genome editing in a wild type genetic background.

178 citations


Cites background from "Ploidy in cyanobacteria"

  • ...Moreover, cyanobacteria maintain multiple copies of their chromosome and numerous rounds of segregation are often necessary to obtain a completely segregated mutant [12]....

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References
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Journal ArticleDOI
TL;DR: Revisions are designed to permit the generic identification of cultures, often difficult through use of the field-based system of phycological classification, and are both constant and readily determinable in cultured material.
Abstract: Summary: On the basis of a comparative study of 178 strains of cyanobacteria, representative of this group of prokaryotes, revised definitions of many genera are proposed. Revisions are designed to permit the generic identification of cultures, often difficult through use of the field-based system of phycological classification. The differential characters proposed are both constant and readily determinable in cultured material. The 22 genera recognized are placed in five sections, each distinguished by a particular pattern of structure and development. Generic descriptions are accompanied by strain histories, brief accounts of strain properties, and illustrations; one or more reference strains are proposed for each genus. The collection on which this analysis was based has been deposited in the American Type Culture Collection, where strains will be listed under the generic designations proposed here.

7,107 citations


Additional excerpts

  • ...elongatus were grown in BG11 medium (Rippka et al., 1979)....

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Journal ArticleDOI
TL;DR: Electron microscopic investigations, together with analytical data on cell wall composition and ribosomal structure, have revealed the common denominators, which are fundamental: bacteria and blue-green algae are the only organisms with cells of the procaryotic type.

2,815 citations


"Ploidy in cyanobacteria" refers background in this paper

  • ...Synechocystis PCC 6803 was isolated 40 years ago and has been cultivated in the laboratory since then (Stanier et al., 1971)....

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  • ...Kunisawa from a freshwater lake in California (Stanier et al., 1971) and deposited at the Pasteur Culture Collection (PCC6803) and the American Type Culture Collection (ATCC 27184)....

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  • ...The species Synechocystis PCC 6803 was isolated from freshwater in California more than 40 years ago (Stanier et al., 1971)....

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Journal ArticleDOI
TL;DR: The advantages and challenges of polyploidy, and its evolutionary potential, are considered.
Abstract: Polyploids — organisms that have multiple sets of chromosomes — are common in certain plant and animal taxa, and can be surprisingly stable. The evidence that has emerged from genome analyses also indicates that many other eukaryotic genomes have a polyploid ancestry, suggesting that both humans and most other eukaryotes have either benefited from or endured polyploidy. Studies of polyploids soon after their formation have revealed genetic and epigenetic interactions between redundant genes. These interactions can be related to the phenotypes and evolutionary fates of polyploids. Here, I consider the advantages and challenges of polyploidy, and its evolutionary potential.

1,882 citations


"Ploidy in cyanobacteria" refers background in this paper

  • ...Many eukaryotic species including ciliates, fish, flowering plants, and even some cell types of humans are polyploid, and advantages as well as disadvantages of polyploidy have been discussed in various reviews (e.g. Wendel, 2000; Osborn et al., 2003; Comai, 2005; Thorpe et al., 2007; Hegarty & Hiscock, 2008)....

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Journal ArticleDOI
TL;DR: Processes and mechanisms of gene and genome evolution in polyploids are reviewed, including the role of transposable elements in structural and regulatory gene evolution; processes and significance of epigenetic silencing; underlying controls of chromosome pairing and mechanisms and functional significance of rapid genome changes are reviewed.
Abstract: Polyploidy is a prominent process in plants and has been significant in the evolutionary history of vertebrates and other eukaryotes. In plants, interdisciplinary approaches combining phylogenetic and molecular genetic perspectives have enhanced our awareness of the myriad genetic interactions made possible by polyploidy. Here, processes and mechanisms of gene and genome evolution in polyploids are reviewed. Genes duplicated by polyploidy may retain their original or similar function, undergo diversification in protein function or regulation, or one copy may become silenced through mutational or epigenetic means. Duplicated genes also may interact through inter-locus recombination, gene conversion, or concerted evolution. Recent experiments have illuminated important processes in polyploids that operate above the organizational level of duplicated genes. These include inter-genomic chromosomal exchanges, saltational, non-Mendelian genomic evolution in nascent polyploids, inter-genomic invasion, and cytonuclear stabilization. Notwithstanding many recent insights, much remains to be learned about many aspects of polyploid evolution, including: the role of transposable elements in structural and regulatory gene evolution; processes and significance of epigenetic silencing; underlying controls of chromosome pairing; mechanisms and functional significance of rapid genome changes; cytonuclear accommodation; and coordination of regulatory factors contributed by two, sometimes divergent progenitor genomes. Continued application of molecular genetic approaches to questions of polyploid genome evolution holds promise for producing lasting insight into processes by which novel genotypes are generated and ultimately into how polyploidy facilitates evolution and adaptation.

1,583 citations

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