Comparison of 432 Pseudomonas strains through integration of genomic, functional, metabolic and expression data

Pseudomonas is a highly versatile genus containing species that can be harmful to humans and plants while others are widely used for bioengineering and bioremediation. We analysed 432 sequenced Pseudomonas strains by integrating results from a large scale functional comparison using protein domains with data from six metabolic models, nearly a thousand transcriptome measurements and four large scale transposon mutagenesis experiments. Through heterogeneous data integration we linked gene essentiality, persistence and expression variability. The pan-genome of Pseudomonas is closed indicating a limited role of horizontal gene transfer in the evolutionary history of this genus. A large fraction of essential genes are highly persistent, still non essential genes represent a considerable fraction of the core-genome. Our results emphasize the power of integrating large scale comparative functional genomics with heterogeneous data for exploring bacterial diversity and versatility

Design and analysis of a tunable synchronized oscillator

BACKGROUND: The use of in silico simulations as a basis for designing artificial biological systems (and experiments to characterize them) is one of the tangible differences between Synthetic Biology and "classical" Genetic Engineering. To this end, synthetic biologists have adopted approaches originating from the traditionally non-biological fields of Nonlinear Dynamics and Systems & Control Theory. However, due to the complex molecular interactions affecting the emergent properties of biological systems, mechanistic descriptions of even the simplest genetic circuits (transcriptional feedback oscillators, bi-stable switches) produced by these methods tend to be either oversimplified, or numerically intractable. More comprehensive and realistic models can be approximated by constructing "toy" genetic circuits that provide the experimenter with some degree of control over the transcriptional dynamics, and allow for experimental set-ups that generate reliable data reflecting the intracellular biochemical state in real time. To this end, we designed two genetic circuits (basic and tunable) capable of exhibiting synchronized oscillatory green fluorescent protein (GFP) expression in small populations of Escherichia coli cells. The functionality of the basic circuit was verified microscopically. High-level visualizations of computational simulations were analyzed to determine whether the reliability and utility of a synchronized transcriptional oscillator could be enhanced by the introduction of chemically inducible repressors. RESULTS: Synchronized oscillations in GFP expression were repeatedly observed in chemically linked sub-populations of cells. Computational simulations predicted that the introduction of independently inducible repressors substantially broaden the range of conditions under which oscillations could occur, in addition to allowing the frequency of the oscillation to be tuned. CONCLUSIONS: The genetic circuits described here may prove to be valuable research tools for the study of synchronized transcriptional feedback loops under a variety of conditions and experimental set-ups. We further demonstrate the benefit of using abstract visualizations to discover subtle non-linear trends in complex dynamic models with large parameter space

Comparison of 432 Pseudomonas strains through integration of genomic, functional, metabolic and expression data

Pseudomonas is a highly versatile genus containing species that can be harmful to humans and plants while others are widely used for bioengineering and bioremediation. We analysed 432 sequenced Pseudomonas strains by integrating results from a large scale functional comparison using protein domains with data from six metabolic models, nearly a thousand transcriptome measurements and four large scale transposon mutagenesis experiments. Through heterogeneous data integration we linked gene essentiality, persistence and expression variability. The pan-genome of Pseudomonas is closed indicating a limited role of horizontal gene transfer in the evolutionary history of this genus. A large fraction of essential genes are highly persistent, still non essential genes represent a considerable fraction of the core-genome. Our results emphasize the power of integrating large scale comparative functional genomics with heterogeneous data for exploring bacterial diversity and versatility

Design and analysis of a tunable synchronized oscillator

BACKGROUND: The use of in silico simulations as a basis for designing artificial biological systems (and experiments to characterize them) is one of the tangible differences between Synthetic Biology and "classical" Genetic Engineering. To this end, synthetic biologists have adopted approaches originating from the traditionally non-biological fields of Nonlinear Dynamics and Systems & Control Theory. However, due to the complex molecular interactions affecting the emergent properties of biological systems, mechanistic descriptions of even the simplest genetic circuits (transcriptional feedback oscillators, bi-stable switches) produced by these methods tend to be either oversimplified, or numerically intractable. More comprehensive and realistic models can be approximated by constructing "toy" genetic circuits that provide the experimenter with some degree of control over the transcriptional dynamics, and allow for experimental set-ups that generate reliable data reflecting the intracellular biochemical state in real time. To this end, we designed two genetic circuits (basic and tunable) capable of exhibiting synchronized oscillatory green fluorescent protein (GFP) expression in small populations of Escherichia coli cells. The functionality of the basic circuit was verified microscopically. High-level visualizations of computational simulations were analyzed to determine whether the reliability and utility of a synchronized transcriptional oscillator could be enhanced by the introduction of chemically inducible repressors. RESULTS: Synchronized oscillations in GFP expression were repeatedly observed in chemically linked sub-populations of cells. Computational simulations predicted that the introduction of independently inducible repressors substantially broaden the range of conditions under which oscillations could occur, in addition to allowing the frequency of the oscillation to be tuned. CONCLUSIONS: The genetic circuits described here may prove to be valuable research tools for the study of synchronized transcriptional feedback loops under a variety of conditions and experimental set-ups. We further demonstrate the benefit of using abstract visualizations to discover subtle non-linear trends in complex dynamic models with large parameter space

Deletion of the <i>aspC</i> gene reduces cell sizes.

<p>(A) Exponentially growing cells in LB or ABTGcasa medium at 37°C were visualised by fluorecence microscopy. (B) Cell length varies with nutrient availability. Bar graphs show average cell lengths in different growth media. Cells in LB, ABTGcasa, ABTG or ABT medium at 37°C and the average size of the cells were measured. More than 100 cells were included in each calculation. The values are average of three individual experiments and standard errors are given as error bars.</p

Rhizopus delemar grown under high and low oxygen conditions

Rhizopus delemar grown under high and low oxygen conditions

Toolkit for Visualization of the Cellular Structure and Organelles in Aspergillus niger

Aspergillus niger is a filamentous fungus that is extensively used in industrial fermentations for protein expression and the production of organic acids. Inherent biosynthetic capabilities, such as the capacity to secrete these biomolecules in high amounts, make A. niger an attractive production host. Although A. niger is renowned for this ability, the knowledge of the molecular components that underlie its production capacity, intercellular trafficking processes and secretion mechanisms is far from complete. Here, we introduce a standardized set of tools, consisting of an N-terminal GFP-actin fusion and codon optimized eforRed chromoprotein. Expression of the GFP-actin construct facilitates visualization of the actin filaments of the cytoskeleton, whereas expression of the chromoprotein construct results in a clearly distinguishable red phenotype. These experimentally validated constructs constitute the first set of standardized A. niger biomarkers, which can be used to study morphology, intercellular trafficking, and secretion phenomena

Toolkit for Visualization of the Cellular Structure and Organelles in Aspergillus niger

Aspergillus niger is a filamentous fungus that is extensively used in industrial fermentations for protein expression and the production of organic acids. Inherent biosynthetic capabilities, such as the capacity to secrete these biomolecules in high amounts, make A. niger an attractive production host. Although A. niger is renowned for this ability, the knowledge of the molecular components that underlie its production capacity, intercellular trafficking processes and secretion mechanisms is far from complete. Here, we introduce a standardized set of tools, consisting of an N-terminal GFP-actin fusion and codon optimized eforRed chromoprotein. Expression of the GFP-actin construct facilitates visualization of the actin filaments of the cytoskeleton, whereas expression of the chromoprotein construct results in a clearly distinguishable red phenotype. These experimentally validated constructs constitute the first set of standardized A. niger biomarkers, which can be used to study morphology, intercellular trafficking, and secretion phenomena