Insertional mutagenesis by random cloning of antibiotic resistance genes into the genome of the cyanobacterium Synechocystis strain PCC 6803

Labarre, Jean; Chauvat, Franck; Thuriaux, Pierre

doi:10.1128/jb.171.6.3449-3457.1989

Cited by 150 publications

(141 citation statements)

References 42 publications

Supporting

Mentioning

134

Contrasting

Unclassified

Order By: Relevance

“…We have demonstrated that vegetative cells of A. ovalisporum are polyploid, with an average genome copy number of 8, similar to that reported for Synechococcus PCC 6301, which ranged between 3 and 18 or Synechocystis PCC 6803 (Labarre et al, 1989). Polyploidy was also reported for Eubacteria such as Thermus thermophilus (4-5 genome copies; Ohtani et al, 2010) and for the halophilic archaeon Halobacterium volcanii with B18 genome copies per cell, a number which was downregulated to 10 genome copies per cell when H. volcanii entered stationary phase (Breuert et al, 2006).…”

Section: Genome and Ribosome Multiplication In Akinetes A Sukenik Et Alsupporting

confidence: 57%

Massive multiplication of genome and ribosomes in dormant cells (akinetes) of Aphanizomenon ovalisporum (Cyanobacteria)

et al. 2011

View full text Add to dashboard Cite

Akinetes are dormancy cells commonly found among filamentous cyanobacteria, many of which are toxic and/or nuisance, bloom-forming species. Development of akinetes from vegetative cells is a process that involves morphological and biochemical modifications. Here, we applied a single-cell approach to quantify genome and ribosome content of akinetes and vegetative cells in Aphanizomenon ovalisporum (Cyanobacteria). Vegetative cells of A. ovalisporum were naturally polyploid and contained, on average, eight genome copies per cell. However, the chromosomal content of akinetes increased up to 450 copies, with an average value of 119 genome copies per akinete, 15-fold higher than that in vegetative cells. On the basis of fluorescence in situ hybridization, with a probe targeting 16S rRNA, and detection with confocal laser scanning microscopy, we conclude that ribosomes accumulated in akinetes to a higher level than that found in vegetative cells. We further present evidence that this massive accumulation of nucleic acids in akinetes is likely supported by phosphate supplied from inorganic polyphosphate bodies that were abundantly present in vegetative cells, but notably absent from akinetes. These results are interpreted in the context of cellular investments for proliferation following a long-term dormancy, as the high nucleic acid content would provide the basis for extended survival, rapid resumption of metabolic activity and cell division upon germination.

show abstract

Section: Genome and Ribosome Multiplication In Akinetes A Sukenik Et Alsupporting

confidence: 57%

Massive multiplication of genome and ribosomes in dormant cells (akinetes) of Aphanizomenon ovalisporum (Cyanobacteria)

et al. 2011

View full text Add to dashboard Cite

show abstract

“…This result indicated that the gene replacement had taken place but that only some of the chromosomes contained the mutation (Synechocystis sp. strain PCC 6803 is a polyploid bacterium containing about 12 chromosomes per cell [29]). Addition of ␣-ketoglutarate to the culture medium did not increase chromosomal segregation (data not shown).…”

Section: Resultsmentioning

confidence: 99%

The NADP+-isocitrate dehydrogenase gene (icd) is nitrogen regulated in cyanobacteria

1996

View full text Add to dashboard Cite

Cyanobacteria are photosynthetic prokaryotes that have an incomplete tricarboxylic acid cycle, lacking ␣-ketoglutarate dehydrogenase and succinyl-coenzyme A synthetase activities (46, 54). The NADP ϩ -isocitrate dehydrogenase (IDH) (EC 1.1.1.42) reaction represents a terminal step in carbon flow; thus, the role of this enzyme is the provision of biosynthetic precursors rather than energy production. The ␣-ketoglutarate produced in the NADP ϩ -IDH reaction is required for ammonium assimilation through the glutamine synthetase-glutamate synthase (GS-GOGAT) pathway (38) and is a key metabolite in the linking of nitrogen and carbon metabolism.IDHs have been purified and characterized from a variety of sources (8); most bacteria have only an NADP ϩ

show abstract

“…Then, the transformation mixture was plated on a 1.5% (wt/vol) agar plate containing BG-11 medium, supplemented with 5 mM glucose, 0.3% (wt/vol) sodium thiosulfate, and 3 g/ml kanamycin. After isolation of individual colonies, segregation of the insert into the multiple copies of the cyanobacterial genome (15,22) was achieved by restreaking the mutants stepwise on plates containing increasing concentrations of kanamycin until complete segregation routinely was accomplished at 20 g/ml kanamycin. Recombinant Taq DNA polymerase (Fermentas) was used for nonpreparative PCR, such as colony PCR, to monitor the segregation process and to verify the correct incorporation of the DNA fragments into the Synechocystis genome.…”

Section: Methodsmentioning

confidence: 99%

Engineering a Cyanobacterial Cell Factory for Production of Lactic Acid

Angermayr

Paszota

Hellingwerf

2012

Appl Environ Microbiol

165

144

View full text Add to dashboard Cite

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.C oncerns about shrinking supplies of fossil fuel and about climate change have been a strong incentive during the past decade for the development of sustainable alternatives to provide fuel and chemical feedstock. Because of these concerns, several different ways to produce solar biofuel have been proposed. Initially the proposed procedures were based on the conversion of agricultural crops into short-chain alcohols. This unfortunately results in a direct competition for resources between the energy sector and food supply. Therefore, second-generation types of processes were proposed, in which one aims at the conversion of the triglyceride fraction from, e.g., green algae into biodiesel, which relaxes the competition between energy and the food supply. Nevertheless, the conversion of CO 2 into biofuel, driven by solar energy, is still rather indirect even in this second-generation approach: CO 2 is first converted into the complex building blocks of biomass, from which subsequently the triglyceride fraction must be extracted and converted into biodiesel.For these reasons, a third-generation type of process has been proposed, in which CO 2 is converted into biofuel directly, accomplished by the introduction of genes encoding a metabolic-e.g., fermentative-pathway into a cyanobacterium, resulting in the emergence of a "photofermentative" chimera. For several biofuel and chemical feedstock products, including ethanol, ethylene, isoprene, D-lactic acid, glucose, sucrose, isobutyraldehyde, and 1-butanol, the proof of principle of this approach has been provided (see references 3,8,10,24,25,28, and 39 and references therein). Ideally, this process would be carried out in such a way that the majority of the CO 2 that is fixed in the cyanobacterial CalvinBenson cycle is converted into the selected product. This implies that the central metabolism in the selected chimera should be largely redirected tow...

show abstract

Insertional mutagenesis by random cloning of antibiotic resistance genes into the genome of the cyanobacterium Synechocystis strain PCC 6803

Cited by 150 publications

References 42 publications

Massive multiplication of genome and ribosomes in dormant cells (akinetes) of Aphanizomenon ovalisporum (Cyanobacteria)

Massive multiplication of genome and ribosomes in dormant cells (akinetes) of Aphanizomenon ovalisporum (Cyanobacteria)

The NADP+-isocitrate dehydrogenase gene (icd) is nitrogen regulated in cyanobacteria

Engineering a Cyanobacterial Cell Factory for Production of Lactic Acid

Contact Info

Product

Resources

About